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Annotators
URL
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dx.doi.org dx.doi.org
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RRID:SCR_01613
DOI: 10.1093/jnci/djae091
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www.sciencedirect.com www.sciencedirect.com
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www.sciencedirect.com www.sciencedirect.com
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AB_3096029
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Local file Local file
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rowdy
adverb ❌. Rowdy is the characteristic that the behavior had. It answers to the question which characteristics do the behavior had not to how was it exhibited.
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accesspharmacy-mhmedical-com.proxy200.nclive.org accesspharmacy-mhmedical-com.proxy200.nclive.org
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does not confer long-lasting protective immunity
no memory
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www.hsph.harvard.edu www.hsph.harvard.edu
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“A good example of an intervention is actually a university,” says Saxena. “If 20 to 30 percent of 20,000 students need support and counseling, and potentially less than 20 percent of them are actually receiving it, we need a five-fold increase in support. If a university health department representative wants to expand mental health care, that person can learn to deliver what is needed in a more effective manner
yes!
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twins
?
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It’s about changing the architecture of care by making care accessible in community settings, making care sensitive to personal needs and priorities, and making sure that people with mental health problems have the same rights as others in the community,
this statement I agree with 100%
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scaling up task-sharing to include other community members (like lay people, family members, colleagues, and workplace administrators) is a crucial step
which would necessitate training and oversight (but I'm not opposed to the concept of 'task-sharing' as I understand it, across professions --but to use a weird analogy, we can't solve the mental health provider crisis by calling everyone a therapist any more than we can solve the national debt by printing off more dollar bills. IMHO.
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and task-sharing
again, unclear to me what is implied or envisioned by 'task-sharing' here.
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pulling mental health out of the health sector
sounds catchy, but what the heck does this mean, exactly? And what are the multitude of implications??
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these functional components
what meaneth you here?
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mild conditions, however, may not need a pricey, in-demand specialist.
agreed, to an extent. I guess it depends on how the term 'specialist' is conceptualized in this article and beyond.
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what most people need are psychological and social interventions that are delivered by non-specialists, people like community health workers, peer support workers, and a range of other general health workers.
hmmm...or greater numbers of TRAINED professionals. A lot of damage is and has been done by those with limited training who step into mental health care in ways that are beyond their capacity. We can't just solve a problem by lowering professional standards and saying, 'well, actually, anyone can do this' - when there is a lot of evidence that calls for greater caution and safeguards to the public than that.
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opentext.uoregon.edu opentext.uoregon.edu
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Logan’s Story
Spacing issue with the poem below.
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opentext.uoregon.edu opentext.uoregon.edu
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saw.
We need a space before bullet points.
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opentext.uoregon.edu opentext.uoregon.edu
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highlight.
Space here before bullet points.
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www.theguardian.com www.theguardian.com
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www.irishtimes.com www.irishtimes.com
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But the climate impact of data centres could be significantly worse than this. Because of the huge strain data centres are placing on power grids, EirGrid placed a de facto moratorium on new connections around Dublin, causing many to seek a connection to the natural gas network to generate electricity on-site.
Wow, it's in Ireland too?!
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Local file Local file
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For twenty ve years, my relationship to writing was equal parts loveand loathing.
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144. See Chris Aldrich’s writings for a comprehensive history of zettelkasten use over the yearsand around the world. https://bo osocko.com/
I love the fact that my personal website is physically the last word in the book and therefore "gets the last word."
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9.5 ere Is No One System
You have to love that one of the final sections of the book is "There is No One System". This gives the reader the confidence to explore and experiment to see what works for them.
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All rights reserved. No part of this book over two hundred y words maybe reproduced, scanned, or distributed in any printed or electronic formwithout permission. Please cite the author and book when quoting.First Edition: June 2024Printed in the United States of AmericaISBN: 979-8-218-45014-4
Doto wastes no time getting into the most important aspects of note taking. Even before the book has begun, the copyright page in the front matter is getting you ready for what is about to come:
Please cite the author and book when quoting.
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Doto, Bob. A System for Writing: How an Unconventional Approach to Note-Making Can Help You Capture Ideas, Think Wildly, and Write Constantly - A Zettelkasten Primer. 1st ed. New Old Traditions, 2024. https://amzn.to/3ztjrfb.
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imperialmathswiki.com imperialmathswiki.com
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finite sub-collections
A previous version of this page contains an erroneous definition of mutual independence of events and sigma-algebra. We apologise for any confusion caused. See the following page for a more thorough discussion: https://imperialmathswiki.com/en/1st_year/MATH40005_Probability_and_Statistics/4#independence
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www.reddit.com www.reddit.com
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The daily cards and journal entries are obviously indexed by chronological date and then within tabbed sections by month and year.
The rest of the other cards with notes are given individual (decimal) numbers and and then are put into numerical order. These numbered cards are then indexed by putting related subject/topic/category words from them onto a separate index card which cross references either a dated card or the numbered card to which it corresponds. These index cards with topical words/phrases are then filed alphabetically into a tabbed alphabetical section (A-Z).
As an example with the card in this post, if I wanted to remember all the books I buy from Octavia's Bookshelf, then I'd create a card titled "Octavia's Bookshelf" and list the title along with the date 2024-08-13 and file it alphabetically within the "O" tab section of the index. Obviously this might be more useful if I had more extensive notes about the book or its purchase on the 2024-08-13 card. I did create a short journal card entry about the bookstore on 08-13 because it was the first time I visited the bookstore in it's new location and decor, so there are some scant notes about my impressions of that which are cross-indexed to that Octavia's Bookshelf card. Thus my Octavia's Bookshelf card has an entry with "The Book Title, 2024-08-13 (J)(R)" where the '(J)' indicates there's a separate journal entry for that day and the '(R)' indicates there's also a receipt filed next to that day's card.
I also created an "Author Card" with the author of the book's name, the title, publication date, etc. I included the purchase date and the reason why I was interested in the book. I'll use that same card to write notes on that particular book as I read it. These author cards are filed in a separate A-Z tabbed 'Bibliography' section for easily finding them as well. (I suppose I could just put them into the primary A-Z index, but I prefer having all the authors/books (I have thousands) in the same section.)
I also have a rolodex section of people filed alphabetically, so I can easily look up Steve and Sonia separately and see what I might have gotten them on prior birthdays as well as notes about potential future gift ideas. I had tickler cards with their names on them filed in early August and now that they're in my to do list, I've moved those cards to August 2025, ready for next year's reminder. Compared to a typical Future Log I don't do nearly as much writing and rewriting when migrating. I just migrate a card forward until it's done or I don't need it anymore.
If you've used a library card index before, the general idea is roughly the same, you're just cross-indexing more than books by title, author and subject. You can index by day, idea, project, or any other thing you like. My card index cabinet is really just a large personal database made out of paper and metal.
The secret isn't to index everything—just the things you either want to remember or know you'll want to look up later and use/re-use.
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imperialmathswiki.com imperialmathswiki.com
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for any finite sub-collections
A previous version of this page contains an erroneous definition of mutual independence of events and sigma-algebra. We apologise for any confusion caused. See the following page for a more thorough discussion: https://imperialmathswiki.com/en/1st_year/MATH40005_Probability_and_Statistics/4#independence
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rsginc.github.io rsginc.github.io
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tbi_data_upcoded_copy <- tbi_data_upcoded
I think you want this
tbi_data_upcoded_copy <- copy(tbi_data_upcoded)
Otherwise tbi_data_upcoded_copy will act like a pointer to tbi_data_upcoded, and not like it's own dataset. If you're in data.table that is.
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www.studentsofhistory.com www.studentsofhistory.com
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Environmental Geography
Read this section on Environmental Geography. Then, add a question you have about the environmental geography of our world.
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Human Geography
Read this section on Human Geography. Then, add a question you have about the human geography of our world.
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Physical Geography
Read this section on Physical Geography. Then, add a question you have about the physical geography of our world.
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Geography is an exciting field of study that takes us on a journey to discover the wonders of our planet.
You will be reading about the three kinds of geography- physical, human and environmental. Be thinking of a question you have aboout each one.
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www.biorxiv.org www.biorxiv.org
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Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
REVIEWER 1
Reviewer #1 Evidence, reproducibility and clarity: Nornes et al. have generated a cohort of arterial enhancers based on in silico analysis and validation with transgenic lines in both zebrafish and mice. They utilized publicly available datasets for chromatin marks, including ATAC-seq on endothelial cells either from cell culture or isolated from mice, as well as EP300 binding, H3K27Ac, and H3K4Me1. Focusing on eight arterial-expressed genes, they identified a putative enhancer region marked by at least one enhancer feature. After validating the activity of these enhancers in zebrafish and mice, the authors assessed the regulatory pathways upstream of these genes. Using ChIP-seq and Cut&Run for key endothelial transcription factors, they discovered that binding sites for SoXF and ETS factors are shared in arterial enhancers, whereas binding sites for Notch, MEF2, and Fox are present only in the subset of identified enhancers. Together this study provides an arterial enhancer atlas that allows further characterisation of regulatory network behind endothelial cell identity.
__Reviewer #1 Major Comment 1: __The authors have assessed 15 enhancers for arterial-venous specificity, by assessing the expression in DA, ISV, cardinal and ventral veins at 2 dpf. Interestingly there is a clear difference in the expression patterns of these enhancers in the zebrafish axial vasculature, especially seen at the level of ISV. The co-localization of the enhancer expression in the endothelium was done using endothelial marks expressed in both venous and arterial EC (kdrl). To fully distinguish if the expression is venous or arterial endothelial compartment colocalization with Tg expressed in arterial (flt1) or venous (lyve1) EC would be informative.
RESPONSE: We agree with the reviewer that a more detailed description of arterial-venous specificity of each enhancer could be included. In the original manuscript, the expression pattern of each enhancer within the vasculature was primarily assessed at 2 days post fertilization (dpf) in Fig 1-2. This identified arteries using direction of blood flow and available descriptive information, as arterial development in 2pdf zebrafish is very stereotypical and already well characterized.
__REVISION (PLANNED): __The original Figure 3A includes a more detailed assessment of arterial-venous specificity at 3dpf for four arterial enhancers (Cxcr4+135, Cxcr4+151, Gja5-78 and Gja5-7, chosen as enhancers representing the four types of expression patterns seen). We will now extend this more detailed analysis to all arterial enhancer:GFP lines. This analysis uses kdrl-mCherry to mark the entire vasculature, comparative to the expression of the arterial enhancers (GFP). This allows us to clearly identify the intersegmental arteries (as opposed to intersegmental veins) by looking for direct connection to the dorsal aorta, and by assessing the direction of blood flow within these vessels. This analysis is done at 3dpf to give time for the intersegmental arteries to acquire identity and connect definitively with the dorsal aorta, and for the diminishment of any GFP expression originating from the initial sprouting from the dorsal aorta. By extending this analysis to the other arterial enhancer zebrafish lines shown in Figure 2, we will be able to more clearly classify the activity of each enhancer within different vascular beds. This information will also be recorded in a new Table better detailing the timing and specificity of activity of each enhancer.
We chose not to use arterial or venous "marker lines" (e.g. Flt1:reporter or Lyve1:reporter) for the simple reason that these are also enhancer:GFP transgenes, and therefore are not necessarily definitive of the arterial or venous lineage per se (e.g. Flt1:GFP expression is controlled by the transcription factors binding the Flt1 enhancer in the same way that Cxcr4+135 and the others are, with the added caveat that the transcriptional regulation of the Flt1 and Lyve enhancers are not well defined). We felt that morphological determination based on direct connections and blood flow direction was therefore more accurate.
__Reviewer #1 Major Comment 2: __In addition, it is striking that cxc4+135 drives the expression in nearly every ISV as cxcl12+269 only every other. Similarly, not all the enhancers are enriched in the DA to the same level. Is there biological significance to this? could authors discuss these results further? The pattern of expression of the unc5b-identified enhancer is also striking, does this reflect the known roles of unc5b in the vascular formation?
__RESPONSE: __We agree, the diversity of enhancer expression patterns within the arterial compartment is notable, and really very interesting. The variations in enhancer expression pattern must be largely influenced by the transcription factor motifs within each enhancer, as these patterns were seen in both transient and stable transgenic zebrafish and therefore largely independent of chromatin integration location.
__REVISION (PLANNED): __The extension of Figure 3A to all enhancer lines (see previous comment) will permit us to more clearly classify the activity of each arterial enhancer within different beds and at different time points. Currently there were no clear links between a particular transcription factor motif/binding and expression pattern, something that is discussed briefly in the original Results and Discussion sections. However, the expansion of Figure 3A to all enhancers, and the creation of a Table summarizing this more systematically will make the link (or lack of one) between expression patterns within the arterial tree and TF motifs easier to appreciate and discuss.
__Reviewer #1 Major Comment 3: __The final part of the paper focuses on defining the presence of "deeply conserved" transcription factor binding sites (TFSB), defined as TFBS that are as conserved as the enhancer sequence surrounding them. In literature, the term 'deep conservation' refers to evolutionary conservation (genomic sequence preservation) in a wide range of species. Therefore, the additional classification presented by the authors based on the surrounding sequence is not clear. As, the KLF motifs in the Ece1in1, which is conserved between mouse and human, are defined as "deeply conserved". However, the FLK motif in the following enhancer, Flk1in10 (one line below), gets classified as non-deeply conserved, despite also being conserved between mouse and human. Thus, in the current form, there is a contradiction in the way the authors use the term 'deeply conserved' and the accepted meaning of this term. To avoid confusion, it would be important to revise this nomenclature.
RESPONSE: We agree that this nomenclature should be revised. Our aim was to develop a standard approach to transcription factor motif analysis that could be applied to enhancers regardless of conservation levels and size, and easily replicated by others. Because not all functional transcription factor motifs within enhancers are necessarily conserved between species, we were careful to label both conserved and non-conserved motifs for each TF examined. Nonetheless, extra emphasis was placed on motifs with confirmatory TF binding evidence (e.g. ChIP-seq/CUT&RUN), and those conserved at the same depth as the surrounding sequence. This was because our previous work on endothelial enhancers clearly indicates that these motifs are far more likely to play a key role in regulation. However, the reviewer is correct to note that referring to such motifs as "deeply" conserved could be misinterpreted.
REVISION (COMPLETED): We have altered our nomenclature. This is explained in the relevant Results sections: "Because the level of conservation of motifs can often be an indication of their importance to enhancer activity, we classified each motif into three categories: strongly conserved (motif conserved to the same depth of the surrounding sequence), weakly conserved (motif conserved in orthologous human enhancer but not to the same depth as the surrounding sequence) and not conserved (motif is not conserved within the orthologous human sequence)".
Two enhancers (Unc5b-57 and Cdh1-1) were only conserved human-mouse, therefore each TF motif within these enhancers could be annotated as both weakly and strongly conserved. As the reviewer noted, this does create confusion. We have now adjusted Figure 5 to use a distinct shape for motifs for which no distinction between weak and strong motif can be made. This does not cover Ece1in1, which is conserved human-mouse-tenrec but was erroneously originally labelled human-mouse only. This error has been corrected.
__Reviewer #1 Minor Comment 1: __Details on how the corresponding non-coding regions between mice and humans were established are missing, what alignment tool was used?
RESPONSE AND REVISION (COMPLETED): This information has now been included in the relevant Results section: "Orthologous human enhancer sequences were identified for every enhancer using the Vertebrate Multiz Alignment & Conservation Track on the UCSC genome browser"
__Reviewer #1 Minor Comment 2: __Not sufficient details are provided for the re-analysis of siRNA data. E.g., which clustering method was used? How the clusters were assigned to cell identities?
RESPONSE AND REVISION (COMPLETED): The details regarding the re-analysis of scRNA data has been expanded in the Methods sections: "Publicly available E12 and E17.5 scRNA-seq data from EC isolated from BmxCreERT2;RosatdTomato lineage traced murine hearts54 was obtained from GEO (GSE214942) prior to processing FASTQ files with the 10X Genomics CellRanger pipeline (V7.0.0). RNA-seq reads were aligned to the mm10 genome reference downloaded from 10X Genomics with the addition of the TdTomato-WPRE sequence. Exclusion of low quality cells with either a UMI count >100,000, total gene count 10%) was performed using Scater55. Data normalisation was performed using the MultiBatchNormalisation method prior to merging of TdTomato positive and negative datasets from individual timepoints. The top 2000 most highly variable genes (excluding mitochondrial and ribosomal genes) in the merged datasets were identified using the Seurat FindVariableFeatures method and utilised to calculate principal component analysis (PCA). Normalised data was scaled using the ScaleData function. Cell clustering was performed using the standard unsupervised graph-based clustering method implemented within Seurat (V4)56. Clusters were visualised in two dimensions using UMAP based non-linear dimensional reduction following the standard Seurat (V4) workflow49. Identified clusters were assigned identities based on marker genes shown to be differentially expressed between populations previously identified in the original study47. Key markers include Npr3 (endocardial), Fabp4 (coronary vascular endothelial), and Nfatc1 (valvular endothelial). The E12.5 sinus venosus EC cluster was assigned based in Aplnr as previously described54. Arterial and venous EC clusters in the E17.5 datasets were annotated based on their enriched expression of Gja5 and Nr2f2, respectively."
__Reviewer #1 Minor Comment 3: __Details about the first HOMER analysis (in the assessment of transcription factor motifs and binding patterns at arterial enhancers) seem to be missing from the methods section.
RESPONSE AND REVISION (COMPLETED): This has been included in the methods: "Analysis of overrepresented motifs within our validated arterial enhancer cohort was performed with HOMER's findMotifsGenome tool using the full validated region of the arterial enhancers. The analysis used the hg38 masked genome and otherwise default settings for all other parameters including randomly selected background regions".
__Reviewer #1 Minor Comment 4: __Pg 12: "For ETS, 23/23 arterial enhancers contained at least one conserved motif (all "deeply" conserved to the same depth as the surrounding enhancer, see S7)". Is it S8, where conservation is indicated?
__ ____RESPONSE AND REVISION (COMPLETED):__ We have corrected this error in the text - no figure actually needed to be referenced here as the previous sentence contained the full list of relevant figures to this statement (Table 2 and Figures 5 and S9, previously called S8, are the places to see this information).
__Reviewer #1 Minor Comment 5: __Figure 1 and 2 for non-zebrafish readers it would be useful to indicate in Figures 1 and 2 the non EC expression that can be observed in the embryos.
RESPONSE AND REVISION (COMPLETED): In addition to arterial expression, a number of the enhancer:GFP transgenes also showed GFP expression within the neural tube. In addition, some transient transgenic embryos also showed ectopic expression in muscle fibres. These have now been indicated on the images in Figure 1 and 2.
__Reviewer #1 Minor Comment 6: __Table S1: Please, indicate in the legend what the asterisk in the H DNAseI column stands for
RESPONSE AND REVISION (COMPLETED): The asterisk indicates where DNaseI hypersensitivity is also seen in multiple non-EC lines. This explanation has been added to the legend.
__Reviewer #1 Minor Comment 7: __Figure S8: The phrasing "conserved to animal" in Figure S8 is misleading. There is no difference in something being conserved to tenrec or manatee, as both are Afrotherians. Hence, the data show that both Efnb2-141 and Ephb4-2 were present in the common ancestor of Afrotherians and humans, namely the ancestor of all placentals. Instead, it would be good to indicate the phylogenetic group for which the presence of the enhancer can be inferred (in this case, Placentalia).
__RESPONSE: __Whilst I appreciate the point, it is the exact sequence that is important here - obviously tenrec and manatee are similar species but still contain differences in nucleotide sequences. The information about conservation leads the reader to the exact species with which the comparison is being made. We tried to restrict this to just one species per phylogenetic group (e.g. tenrec, opossum, chicken, zebrafish) but occasionally this was not possible.
Reviewer #1 Significance
To date, a systematic approach to identifying the regulatory networks driving endothelial cell identity is missing. This study provides important datasets and validation of enhancers involved in arterial gene expression and the associated transcription factors. Although this is only the tip of the iceberg, this work represents a significant milestone in the systematic understanding of how arterial gene expression is regulated. Overall, this study offers a powerful resource for understanding arterial gene regulation and conducting genome-wide studies of arterial enhancers.
__RESPONSE: __We thank the reviewer for these kind words. Whilst we agree this is only a very small snapshot of all the arterial enhancers involved in gene regulation, we would like to stress that not only is this a massive increase to what has been known previously, but is also deliberately focused on the genes used to define arterial identity during development and in the adult, therefore these enhancers by themselves form an extremely valuable dataset with which to study the key factors driving arterial differentiation and identity.
__ __
REVIEWER 2
__Reviewer #2 Evidence, reproducibility and clarity: __In this work, Nornes and collaborators have described a cohort of arterial enhancers that drive gene expression in arteries and not in veins. The paper is very well written and it is very informative. The authors have used in silico models to identified the potential artery enhancers and then used different developmental in vivo systems, zebrafish and mice, to validate their findings. Finally, the authors have explored what transcription factors may be binding the identified enhancer sequences and thus, drive arterial gene expression. I would like to congratulate the authors for this work that it has been a pleasure to read and review.
Reviewer #2 Major Comment 1: In their identification of enhancers, the authors consider a candidate every enhancer that has a putative mark in both mouse and human. Nevertheless, all the human data comes from in vitro analysis. Considering how much cell culture affects endothelial cell identity, inducing effects like EndoMT, would this have any effect on the enhancer selection? Would it be possible to search any human in vivo data? Would this allow for even stronger and more relevant sequences?
__RESPONSE: __We agree that the use of human endothelial cells in culture raises some potential issues. However, we stress that the mouse EC enhancer marks, which played a key role in defining putative enhancers, come from in vivo analysis (E11 embryos, P6 retina and adult aorta), limiting the potential for significant impact from cell culture-induced issues. Whilst we would have enthusiastically incorporated human in vivo data had it been available, our approach was still indisputably successful at identifying arterial enriched/specific enhancers.
We consider it unlikely that culture/identity-related problems with human cultured ECs led to a significant undercount of enhancers, in part because comparatively few regions with enhancer marks in mouse in vivo ECs were excluded due to the absence of human enhancer marks. In fact, Cxcr4, Cxcl12, and Gja5 were poorly transcribed in the human cell lines studied here and consequently only enhancer marks in mouse were used to define putative enhancers for these three genes (this is clearly stated in the Results section). If a similar rational had applied to the remaining five genes, only an additional six putative enhancers would have been tested (one for Gja4, two for Nrp1 and three for Unc5b). However, we felt it made sense to include analysis of human enhancer marks for these five genes, as all were expressed in the human ECs used (as indicated by H3K1Me3 and DNaseI hypersensitivity at promoter regions) and orthologous human enhancers were identified for all. Additionally, our retrospective analysis of previously described mammalian in vivo-validated EC enhancers (Table S1 in the original manuscription, including eight arterial enhancers) found that all 32 were marked by at least one enhancer mark in human samples (1/32 did not contain mouse enhancer marks). We also tested eleven regions that did not reach our putative enhancer threshold, including five with only mouse marks. None of these directed expression in transgenic analysis.
Reviewer #2 Major Comment 2: The human data comes from vein endothelial or microvasculature endothelial cells. Specially because some of the enhancers identified by the authors drive also vein expression, could the authors discriminate whether this is due to the identification coming from vein cells. Is there available data from HAECs? Would this not be conceptually more correct that using vein endothelial cells data? This should be at least discussed in the paper.
__RESPONSE AND REVISION (COMPLETED): __We have now included a comparison with enhancer marks from HAECs, telo-HAECs and HUAECs as a new Figure S5. The enhancer marks seen in these cells were very similar to those in the HUVEC and microvascular cells already surveyed. Had enhancer marks within HAECs/telo-HAEC/HUAECs been included as a human enhancer mark in our initial survey, it would have been unlikely to have altered our analysis, although we agree it would have made it more conceptually correct. We chose not to go back and engineer this into our original enhancer selection rational however as we felt it would be intellectually dishonest. A paragraph has been added to the Results section about this analysis.
Reviewer #2 Major Comment 3: Although the authors use the mouse embryo to further validate their finding beyond the zebrafish, the expression are a bit different. While on the fish the enhancers label smaller vessels of arterial identity, in the mouse, only bigger arteries are marked. Is this defined by the time of the analysis?
__RESPONSE: __This experiment was conducted to demonstrate that these enhancers were arterial enriched in both zebrafish and mouse transgenesis, and feel this is clearly shown by the current data. Whilst I do not really agree that the expression pattern is different (for example, the Gja5 enhancers are more restricted to the major arteries in both zebrafish and mouse, compared to the more widely expressed Efnb2-333), this is challenging to ascertain at a single time-point in a transient transgenic mouse assay. Whilst it would be potentially interesting to better assess the activity of these enhancers over time in mice, we consider this a lengthy experiment (multiple stable lines would need to be established and characterized for each enhancer) which would not add particular benefit to this paper.
Reviewer #2 Major Comment 4: The analysis of the enhancers is only done during development. Is the activity of these enhancers maintained through live or only important for artery vs vein determination? Is the expression of the different enhancer reporters maintained into adulthood?
RESPONSE AND REVISION (PLANNED): We agree this would be interesting to ascertain. We plan to examine the activity of enhancer:GFP activity in adult fish fins (which are accessible even without crossing into a casper background, which is beyond the timescale of this project) in the fully revised version of this paper. We have already conducted a feasibility study on four arterial enhancers:GFP lines (Gja5-7:GFP, Gja5-78:GFP, Gja4+40:GFP and Efnb2-333:GFP), which found that all four were still active, and arterial-specific, in the adult.
Reviewer #2 Significance
This is a very well done study with potential interest for vascular biologists, in particular to those interested in the determination between artery and veins in a context of development. It advances our knowledge on the field of vascular biology as it not only proposes potential enhancers but also goes on to validation of the enhancers. Nevertheless, it is important to note that some of this enhancers have been identified from in vitro human data. In vitro culture of endothelial cells affects their cellular identity and thus, this study may have underscored many potential enhancers.
REVIEWER 3
__Reviewer #3: Evidence, reproducibility and clarity: __This manuscript by Nornes et al analyzed multiple published databases and identified a group of putative enhancers for 8 selected non-Notch arterial genes in mouse and human ECs. These enhancers were cloned and screened in fish embryos to test their effect in driving GFP reporter expression, which narrowed down a cohort of enhancers for further testing of expression activities in mouse embryonic arteries. The authors then analyzed the sequences of these enhancers, and identified binding motifs of ETS, SOX-F, FOX and MEF2 family TFs and Notch transcription regulator RBPJ commonly present in closed proximity in these arterial enhancers, suggesting interaction between these TFs in determination of arterial identity.
Reviewer #3 Major Comment : This study provides an enormous amount of bioinformatic data analysis and screening results in transgenic fish and mouse models, which led to the discovery of a group of arterial enhancers and TFs binding motifs essential in regulating arterial identity.
Reviewer #3 Other Comments ____1: Choice of arterial genes is slightly biased. Acvrl1/Alk1 is not enriched in arterial ECs. Sema3G, which is highly expressed in arterial ECs, is missing. UNC5B is enriched in arterial ECs but also expressed by sprouting ECs (PMID: 38866944).
__RESPONSE: __When we started this project, scRNA-seq datasets in the developing vasculature were less available. Consequently, we initially based our choice of genes on data from Raftrey et al., Circ Res 2021 (available earlier on bioRxiv), which was focused on mouse coronary arterial ECs at the timepoints that arteries differentiate. This found Acvrl1 to be arterial enriched (not a novel observation, many publications treat Acvrl1 as arterial specific or arterial-enriched) and did not list Sema3g. We also considered a wider dataset from mouse and human mid-gestation embryos when available (Hou et al., Cell Research 2022). However, it is important to note that we did not aim to investigate every arterial-enriched gene, rather to use these datasets to help identify loci associated with gene expression patterns which indicated a high likelihood of containing arterial enhancers active during arterial differentiation.
Sc-RNAseq data from both Raftery et al., and Hou et al., indicated that arterial ECs are subdivided into two groups, reflecting maturity but also potentially slightly different developmental trajectories. The genes studied here were therefore selected to evenly cover both subgroups, with Acvrl1, Cxcl12, Gja5 and Nrp1 primarily restricted to the mature arterial EC subgroup, while Cxcr4, Efnb2, Gja4 and Unc5b were also expressed in the less mature/arterial plexus/pre-arterial EC subgroup. It is notable that genes within the latter subgroup are also associated with angiogenic/sprouting ECs (Dll4 also belongs to this subgroup), which likely indicates biological links between angiogenesis and arterial identity rather than a problem in gene choice and specificity.
__REVISION (COMPLETED): __This is already discussed in the Results section (angiogenic expression of arterial genes is discussed within the MEF2 and RBPJ sections) and in the Discussion (paragraph 2, referring to different expression patterns within arterial ECs). However, we have now edited the relevant Results section to better explain gene selection: "It is therefore clear that a better understanding of the regulatory pathways directing arterial differentiation requires the identification and characterization of a larger number of arterial enhancers directing the expression of key arterial identity genes. To identify a cohort of such enhancers, we looked in the loci of eight non-Notch genes: Acvrl1(ALK1) Cxcr4, Cxcl12, Efnb2, Gja4(CX37), Gja5 (CX40), Nrp1 and Unc5b. Although not a definitive list of arterial identity genes, single cell transcriptomic analysis indicates these genes are all significantly enriched in arterial ECs4,20, and are commonly used to define arterial EC populations in mouse and human scRNAseq analysis4,5,20,54. Additionally, single-cell transcriptomic data indicates that arterial ECs can be divided into two subgroups4,20. The genes selected here are equally split between subgroups (Acvrl1, Cxcl12, Gja5 and Nrp1 from the mature arterial EC subgroup, Cxcr4, Efnb2, Gja4 and Unc5b from the less mature/arterial plexus/pre-arterial EC subgroup)4,20. We did not exclude genes also implicated in angiogenesis/expressed in sprouting ECs, as these genes formed that vast majority of those associated with the less mature EC subgroup".
Reviewer #3 Other Comments ____2: Exclusion of Notch genes. Although the reason for choosing non-notch genes and excluding notch genes for screening is addressed in this paper, it would be interesting to examine how the arterial enhancers identified in this study are present in the Notch genes, especially Dll4 (enriched in arterial and sprouting ECs) and Jag1 (enriched in arterial ECs).
__RESPONSE: __Previous work from our lab and others has already examined arterial enhancers for Notch pathway genes. We already included these enhancers in all our later analysis (Figure 5-6 and relevant supplemental figures), including analysis of TF motifs.
Reviewer #3 Other Comments ____3: SoxF family TFs. Among the 3 members of SoxF TFs, only Sox17 and Sox7 were assessed. Though not specific, Sox18 is highly expressed in the arteries. On the contrary, Sox7 is highly expressed in the vein and shows weak expression in arterial ECs (PMID: 26630461).
__RESPONSE AND REVISION (PLANNED): __We agree. We will include assessment of SOX18 binding in our final revised manuscript. An antibody for this analysis has been identified already.
Reviewer #3 Other Comments ____4: Minor inaccuracy in Intro/paragraph 3: though sox17 is reported as indispensable for arterial specification (PMID: 24153254), losing a single SoxF factor does not seem to completely compromise the arterial program (PMID: 24153254, PMID: 26630461). A combined loss of Sox17/18, or Sox 7/17/18, seems to do the job (PMID: 26630461).
__RESPONSE: __We have altered this section: "The evidence linking SOXF transcription factors to arterial differentiation is more extensive, with loss of either SOX17 (the SOXF factor most specific to arterial ECs) or SOX7 resulting in arterial defects21-24. Whilst losing a single SOXF factor does not entirely compromise the arterial program, arterial differentiation appears absent after compound Sox17;Sox18 and Sox7;Sox17;Sox18 deletion, although this occurs alongside significantly impaired angiogenesis and severe vascular hyperplasia21-24. PMID 24153254 is reference 23, PMID 26630461 is reference 24.
Reviewer #3 Other Comments ____5: Fig.4 e14.5 mouse embryos. If the observation aims to assess the dorsal aorta, it would be better to use mouse embryos at mid-gestation (e9.5-10.5), when the paired DAs are formed with arterial identity but haven't been remodelled and fused as one single aorta. The morphological data in this figure would be better to show the colocalization of LacZ expression and an arterial marker (e.g. Sox17) using immulfluorescence staining instead of purely lacZ.
RESPONSE: This experiment was primarily conducted to demonstrate that our enhancers were arterial enriched in both zebrafish and mouse transgenesis, and feel this is clearly shown with the e14.5 transgenic embryos originally shown. We chose e14.5 because it matched the timepoints used for the single cell transcriptomics first used to select the target arterial identity genes, and feel it is a good match to 2-3 dpf zebrafish in terms of arterial differentiation mechanisms. We agree that E9-10 would have also been an additional useful timepoint, but we do not have the resources to generate this data nor consider it essential for the conclusions of our work here.
__REVISION (PLANNED): __We are unable to perform immunofluorescence in the e14.5 transgenic embryos due to the fixation and staining solutions used for X-gal staining (which was done by an external company and could not be altered), but agree additional information is needed to demonstrate arterial endothelial specificity. We will therefore expand the analysis of sectioned embryos (currently restricted to just the Efnb2-333:LacZ transgene) to all enhancers shown in Figure 4. This analysis has some limitations due to infiltration of the X-gal solution to deeper tissues, but is anticipated it will clearly show enhancer activity in arterial endothelial cells rather than venous ECs or smooth muscle cells.
__Reviewer #3 (Significance (Required)): __This novel work establishes an important foundation for future understanding of how TFs may interact to determine arterial specification.
Other revisions
In addition to changes suggested by the reviewers, we also made one additional adjustment to the paper to include analysis of two additional putative enhancers (Efnb2-159 and Cxcr4+119). These were initially omitted in error yet both regions reach the standard of testable putative enhancers (noted in small changes to Figure S1 and Table S2). When tested in zebrafish transient transgenic embryos, Cxcr4+119 was inactive whilst Efnb2-159 was active in arterial endothelial cells. The relevant tables and figures have been adjusted to reflect these changes, the most significant of which are the inclusion of Efnb2-159 positive zebrafish in Figure 1 (and the necessity to create an additional supplemental Figure (S3) to accommodate the increased number of images), and analysis of Efnb2-159 transcription factor motifs/binding as part of Figure 5 and 6. No conclusions were altered by the inclusion of this additional data.
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Referee #3
Evidence, reproducibility and clarity
This manuscript by Nornes et al analyzed multiple published databases and identified a group of putative enhancers for 8 selected non-Notch arterial genes in mouse and human ECs. These enhancers were cloned and screened in fish embryos to test their effect in driving GFP reporter expression, which narrowed down a cohort of enhancers for further testing of expression activities in mouse embryonic arteries. The authors then analyzed the sequences of these enhancers, and identified binding motifs of ETS, SOX-F, FOX and MEF2 family TFs and Notch transcription regulator RBPJ commonly present in closed proximity in these arterial enhancers, suggesting interaction between these TFs in determination of arterial identity.
Major comments:
This study provides an enormous amount of bioinformatic data analysis and screening results in transgenic fish and mouse models, which led to the discovery of a group of arterial enhancers and TFs binding motifs essential in regulating arterial identity.
Other comments:
- Choice of arterial genes is slightly biased. Acvrl1/Alk1 is not enriched in arterial ECs. Sema3G, which is highly expressed in arterial ECs, is missing. UNC5B is enriched in arterial ECs but also expressed by sprouting ECs (PMID: 38866944).
- Exclusion of Notch genes. Although the reason for choosing non-notch genes and excluding notch genes for screening is addressed in this paper, it would be interesting to examine how the arterial enhancers identified in this study are present in the Notch genes, especially Dll4 (enriched in arterial and sprouting ECs) and Jag1 (enriched in arterial ECs).
- SoxF family TFs. Among the 3 members of SoxF TFs, only Sox17 and Sox7 were assessed. Though not specific, Sox18 is highly expressed in the arteries. On the contrary, Sox7 is highly expressed in the vein and shows weak expression in arterial ECs (PMID: 26630461). Minor inaccuracy in Intro/paragraph 3: though sox17 is reported as indispensable for arterial specification (PMID: 24153254), losing a single SoxF factor does not seem to completely compromise the arterial program (PMID: 24153254, PMID: 26630461). A combined loss of Sox17/18, or Sox 7/17/18, seems to do the job (PMID: 26630461).
- Fig.4 e14.5 mouse embryos. If the observation aims to assess the dorsal aorta, it would be better to use mouse embryos at mid-gestation (e9.5-10.5), when the paired DAs are formed with arterial identity but haven't been remodeled and fused as one single aorta. The morphological data in this figure would be better to show the colocalization of LacZ expression and an arterial marker (e.g. Sox17) using immulfluorescence staining instead of purely lacZ.
Significance
This novel work establishes an important foundation for future understanding of how TFs may interact to determine arterial specification.
-
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Referee #2
Evidence, reproducibility and clarity
In this work, Nornes and collaborators have described a cohort of arterial enhancers that drive gene expression in arteries and not in veins. The paper is very well written and it is very informative. The authors have used in silico models to identified the potential artery enhancers and then used different developmental in vivo systems, zebrafish and mice, to validate their findings. Finally, the authors have explored what transcription factors may be binding the identified enhancer sequences and thus, drive arterial gene expression. I would like to congratulate the authors for this work that it has been a pleasure to read and review.
Major comments:
- In their identification of enhancers, the authors consider a candidate every enhancer that has a putative mark in both mouse and human. Nevertheless, all the human data comes from in vitro analysis. Considering how much cell culture affects endothelial cell identity, inducing effects like EndoMT, would this have any effect on the enhancer selection? Would it be possible to search any human in vivo data? Would this allow for even stronger and more relevant sequences?
- The human data comes from vein endothelial or microvasculature endothelial cells. Specially because some of the enhancers identified by the authors drive also vein expression, could the authors discriminate whether this is due to the identification coming from vein cells. Is there available data from HAECs? Would this not be conceptually more correct that using vein endothelial cells data? This should be at least discussed in the paper.
- Although the authors use the mouse embryo to further validate their finding beyond the zebrafish, the expression are a bit different. While on the fish the enhancers label smaller vessels of arterial identity, in the mouse, only bigger arteries are marked. Is this defined by the time of the analysis?
- The analysis of the enhancers is only done during development. Is the activity of these enhancers maintained through live or only important for artery vs vein determination? Is the expression of the different enhancer reporters maintained into adulthood?
Significance
This is a very well done study with potential interest for vascular biologists, in particular to those interested in the determination between artery and veins in a context of development. It advances our knowledge on the field of vascular biology as it not only proposes potential enhancers but also goes on to validation of the enhancers. Nevertheless, it is important to note that some of this enhancers have been identified from in vitro human data. In vitro culture of endothelial cells affects their cellular identity and thus, this study may have underscored many potential enhancers.
-
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Referee #1
Evidence, reproducibility and clarity
Summary:
Nornes et al. have generated a cohort of arterial enhancers based on in silico analysis and validation with transgenic lines in both zebrafish and mice. They utilized publicly available datasets for chromatin marks, including ATAC-seq on endothelial cells either from cell culture or isolated from mice, as well as EP300 binding, H3K27Ac, and H3K4Me1. Focusing on eight arterial-expressed genes, they identified a putative enhancer region marked by at least one enhancer feature. After validating the activity of these enhancers in zebrafish and mice, the authors assessed the regulatory pathways upstream of these genes. Using ChIP-seq and Cut&Run for key endothelial transcription factors, they discovered that binding sites for SoXF and ETS factors are shared in arterial enhancers, whereas binding sites for Notch, MEF2, and Fox are present only in the subset of identified enhancers. Together this study provides an arterial enhancer atlas that allows further characterisation of regulatory network behind endothelial cell identity.
Major comments:
The authors have assessed 15 enhancers for arterial-venous specificity, by assessing the expression in DA, ISV, cardinal and ventral veins at 2 dpf. Interestingly there is a clear difference in the expression patterns of these enhancers in the zebrafish axial vasculature, especially seen at the level of ISV. The co-localization of the enhancer expression in the endothelium was done using endothelial marks expressed in both venous and arterial EC (kdrl). To fully distinguish if the expression is venous or arterial endothelial compartment colocalization with Tg expressed in arterial (flt1) or venous (lyve1) EC would be informative. In addition, it is striking that cxc4+135 drives the expression in nearly every ISV as cxcl12+269 only every other. Similarly, not all the enhancers are enriched in the DA to the same level. Is there biological significance to this? could authors discuss these results further? The pattern of expression of the unc5b-identified enhancer is also striking, does this reflect the known roles of unc5b in the vascular formation? The final part of the paper focuses on defining the presence of "deeply conserved" transcription factor binding sites (TFSB), defined as TFBS that are as conserved as the enhancer sequence surrounding them. In literature, the term 'deep conservation' refers to evolutionary conservation (genomic sequence preservation) in a wide range of species. Therefore, the additional classification presented by the authors based on the surrounding sequence is not clear. As, the KLF motifs in the Ece1in1, which is conserved between mouse and human, are defined as "deeply conserved". However, the FLK motif in the following enhancer, Flk1in10 (one line below), gets classified as non-deeply conserved, despite also being conserved between mouse and human. Thus, in the current form, there is a contradiction in the way the authors use the term 'deeply conserved' and the accepted meaning of this term. To avoid confusion, it would be important to revise this nomenclature.
Minor:
Details on how the corresponding non-coding regions between mice and humans were established are missing, what alignment tool was used?
Not sufficient details are provided for the re-analysis of siRNA data. E.g., which clustering method was used? How the clusters were assigned to cell identities?
Details about the first HOMER analysis (in the assessment of transcription factor motifs and binding patterns at arterial enhancers) seem to be missing from the methods section.
Pg 12: "For ETS, 23/23 arterial enhancers contained at least one conserved motif (all "deeply" conserved to the same depth as the surrounding enhancer, see S7)". Is it S8, where conservation is indicated?
Figure 1 and 2 for non-zebrafish readers it would be useful to indicate in Figures 1 and 2 the non EC expression that can be observed in the embryos.
Table S1: Please, indicate in the legend what the asterisk in the H DNAseI column stands for
Figure S8: The phrasing "conserved to animal" in Figure S8 is misleading. There is no difference in something being conserved to tenrec or manatee, as both are Afrotherians. Hence, the data show that both Efnb2-141 and Ephb4-2 were present in the common ancestor of Afrotherians and humans, namely the ancestor of all placentals. Instead, it would be good to indicate the phylogenetic group for which the presence of the enhancer can be inferred (in this case, Placentalia).
Significance
To date, a systematic approach to identifying the regulatory networks driving endothelial cell identity is missing. This study provides important datasets and validation of enhancers involved in arterial gene expression and the associated transcription factors. Although this is only the tip of the iceberg, this work represents a significant milestone in the systematic understanding of how arterial gene expression is regulated. Overall, this study offers a powerful resource for understanding arterial gene regulation and conducting genome-wide studies of arterial enhancers.
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seven more on the way
Perkins (Dorchester), Mildred Ave (Mattapan), Madison Park (Roxbury), Mattahunt (Mattapan), Marshall (Dorchester), Draper (West Roxbury), Clougherty (Charlestown).
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demo.connect.dev.volker.network demo.connect.dev.volker.network
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Have a registration code?
This is only if the user doesn't follow the hyperlinked button. I think it should be moved to the bottom of sign in or be in a troubleshooting flow only
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gdgoenkadelhincr.blogspot.com gdgoenkadelhincr.blogspot.com
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In kindergarten, encouragement creativity is key to developing young minds. One effective way to do this is through 'Write the Room' kindergarten school in Faridabad. These centers transform classroom spaces into interactive learning environments where children can explore, write, and engage with their surroundings. This blog post delves into the benefits, setup, and tips for maximizing the impact of 'Write the Room' centers in your kindergarten classroom.
Teachers can adapt 'Write the Room' to suit various themes, such as seasons, holidays, or subject-specific vocabulary. By integrating this activity into daily routines, educators can create a lively and engaging classroom atmosphere that promotes both physical and cognitive development.
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www.wikihow.com www.wikihow.com
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How to Get Dimples Naturally
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developer.mozilla.org developer.mozilla.org
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View Transitions API
yes
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What Is My Favorite Color?
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10 Easy Ways to Get Free Clothes From Shein & Score Huge Discounts
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www.thediff.co www.thediff.co
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It is, again, very hard to blame capitalism for something that capitalists love to complain about
No, it's quite easy - capital is the subject motivating externality-producing actions. absolute buffoon of a take.
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There are plenty of people who would do these jobs, at their current pay, if that were an option, so the ability to paralyze a city like this is a function of unions, not of the job itself.
incoherent separation between the social facticity of the "union" and the "job" - jobs are unionized because social forces mote them be so. author is ignorant of the very idea of a "social force" beyond individual choice.
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That parent organization is the German military. Do these rules come from a company arbitrarily trying to inconvenience its customers, or from an unaccountable rules-maker somewhere in the German government?
an organization without which the institution of private property is impossible. try again.
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This idea that rich people create fake jobs in order to have an impressive-looking number of economic dependents runs into a few other problems
simplistic means-ends reduction of Graeber's work. farcical drivel.
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That doesn't make their jobs fake. It just makes them jobs, i.e. the kind of thing you have to do for money because the things that you enjoy doing get done for free
dumbass has no idea what Graeber means by bullshit
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r.campaign.optimal-systems.de r.campaign.optimal-systems.de
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Von unserem Partner NTT Data
Wir sind hier übrigens auch! Daher bitte auch den Hinweis auf OS:
https://www.optimal-systems.de/termine/transformation-now-2024?_gl=1wylmhu_upMQ.._gaMTgyNzc4MjM5Mi4xNzIzNjUwNzQ3_ga_4CCG3SLXEX*MTcyMzY1MDc0Ny4xLjAuMTcyMzY1MDc0Ny4wLjAuMA..
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Bestandskundentag
Interessententag!
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Preisliste
Welcher?
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als jederzeit
dient jederzeit als
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nach der international anerkannten Norm ISO
irgendwie ist das Doppelt, aber du brauchst Zeichen, damit es gleich lang ist? Denn ISO steht ja für International (!) Organization for Standardization bzw. Normung. Vielleicht einfach noch so was wie: "weltweit erfolgreichste und bekannteste Managementsystem für Qualitätsmanagement" verwenden
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Poweruser
Ist das wirklich ein einzelnes Wort oder Power User
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Managerin
Auf der Blogseite steht Manager? Anpasssen?
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?
Unten auf dem Butten vielleicht eher "Zum Thema"
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zur richtigen Zeit die richtigen Dinge
ich kenne es als "die richtigen Dinge zur richtigen Zeit". Ist ein kleiner Unterschied. Aber mit deiner Wahl ist der Fokus auf die Zeit mit den Workflows besser.
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asteriskmag.com asteriskmag.com
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AI companies fudge their benchmarks to sway consumers
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philip-ciwf.medium.com philip-ciwf.medium.com
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- Species extinction rate is up to 1,000 times higher than the past ten million years.
- Insect populations, once abundant, have significantly declined.
- Bee numbers have halved in the past 25 years; crucial for pollination.
- Lapwing birds are the most rapidly declining species in Europe.
- Biodiversity loss is driven by agricultural industrialization.
- The UK is one of the most nature-depleted countries globally.
- Urgent need to address the crisis to preserve ecosystems for future generations.
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www.kaieteurnewsonline.com www.kaieteurnewsonline.com
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Ask the Minister…who would read it? Glenn Lall? I can’t even go through that. You need technical people. A Field Development Plan for an oil and gas sector you need specialized people. They would put Glenn Lall, the specialist to read it. Who in your agency would read it if you see it?
I think this justification is bullshit.
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www.sciencedirect.com www.sciencedirect.com
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Introduction
Test Annotation.
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www.theguardian.com www.theguardian.com
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www.wikihow.com www.wikihow.com
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How to Tie a Dimple
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www.youtube.com www.youtube.com
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there is one thing that I want to to do on top of proving you know or disproving fact falsifying or not this theory is to finding ways in which people that are ready can have an extraordinary experience of Consciousness like did not through drugs but through methods you know way to breathe or different ways of special meditations what have you they are sufficiently welld developed that they can help the process of people experiencing themselves their Unity with one
for - Federico Faggin - high priority objective - find and implement ways to catalyze authentic awakening experiences for those who are ready
Federico Faggin - high priority objective - find and implement ways to catalyze authentic awakening experiences for those who are ready - Deep Humanity BEing journeys!
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I want to figure out find out help find out ways in which we can have things where maybe at the most you need to dedicate a week of your life you know because you need to be in a special environment in order to have the the sort of the the conditions in which this can happen and can have those experiences and if say 30% of the people that claim to be ready actually have one of those experien that would be a marvelous objective to reach so that's what I'm thinking right now
for - Federico Faggin - high priority objective - find and implement ways to catalyze authentic awakening experiences in a short time - ie - one week
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to me the first step for being able to grow as a human being and as a true human being and express our true nature is to takeing first responsibility for what happens in our life good and bad and the next step is to be honest about yourself so the honesty was to recognize that I was unhappy and I was pretending to be happy so I recognize what normally people do not because they don't want to change their belief and so they continue to be unhappy
for - answer - how to experience nondual - how to experience non-separation and the authentic self - Federico Faggin
answer - how to experience nondual - how to experience non-separation and the authentic self - Be sincere in acknowledging your unhappiness and - take responsibility for it - Be a sincere seeker - The intensity of your search is like a prayer
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he Experience you had when you felt this beam coming out of you uh what type of experiences should people or could people aim in order to get access to this sort of information do they need some sort of a psychedelic do they need to meditate they need to read the WR books
for - question - how to experience nondual - how to experience non-separation
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that's why the computer can never be conscious because basically he has none of the characteristics of qualia and he certainly doesn't have free will and Free Will and conscious must work together to create these fields that actually can can direct their own experience and create self-conscious entities from the very beginning
for - AI - consciousness - not possible - Frederico Faggin
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with six postulates that are purely informational you can derive all the basic equations of physics and so that's a major piece of work because it demonstrated the intuition of John Wheeler that in 1995 said the famous it from bit so wheeler into it that matter is actually most likely produced by information
for - quantum physics - John Wheeler's theory - validating
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and so so that theory was born by my effort also to try to figure out how do I connect what we all this Rich knowledge that we have about the physical world in physics with this inner world that I knew from the inside and that was called operational probabilistic Theory
for - CIP OPT integration - Federico Faggin
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the second book irreducible you have many quotes at the start of each CH chapter and and it's kind of incredible when you realize how many physicists back in the day like Schrodinger Max plank all these people have these amazing quotes on Consciousness being such a fundamental aspect of reality
for - consciousness - primacy of in physics - quotations from famous scientists
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this is the essence also of one and if we are part all well then we all can have this experience because it is who we are
for - democracy of the sacred - illusion of Maya - poverty mentality
democracy of - the sacred - illusion of Maya - Theoretically, we should all be able to awaken to the sacred, because THAT is what we all are! - And yet, most of us are so deluded that we cannot access that experience - Maya's illusion of separation is so strong - Poverty mentality is so strong
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t was so profound and so deeply felt to be true it was a direct experience of Consciousness that I never had before and it revealed that I am the totality of reality observing itself from a one point of view
for - quote - awakening experience - Federico Faggin
quote - awakening experience - Federico Faggin - (see below)
- What I was observing was energy that previously had come out of my chest and
- It was physical energy
- It was not an imagination
- It was physical energy was
- It was a white light that
- It felt like a love that I never felt before and
- It was love, joy and peace
- I never I never had experienced peace before
- It was like like that's me this is my home this is this is who - I am that energy then now exploded now is everywhere and now I am, my consciousness is in that energy
- My feelings are in that consciousness, which is also outside inside your body and o
- Outside your body is everywhere well that experience can change your idea of who you are very quickly because
- Apart from the fact that
- it was so profound and
- so deeply felt to be true
- it was a direct experience of Consciousness that I never had before and
- it revealed that I am the totality of reality observing itself from a one point of view
- Apart from the fact that
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I had extraordinary experience of Consciousness which is written in the book uh in the in fact both books that I that I uh printed where essentially I experienc myself as the Observer and the observe but I retain my point of view I was observing the world that and the world was me because my conscious was in that world that I was observing but I was observing
for - epoche - kensho - satori - awakening experience - Federico Faggin
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I was betrayed by physicalism
for - hard problem of consciousness - Federico Faggin
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a big part of the book and a big part of your previous book as I've read both of them is your joury because you describe your life going into different phases
for - Federico Faggin - personal journey - profound awakening experience - reorientation of consciousness - from materialist - to idealist
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when the body dies you are gone because you are the body in this other theory on the other hand we are the field that controls the body so when the drone dies don't go anywhere you stay where you were and you continue to live
for - comparison of death in - material vs idealist theories
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Consciousness is the perfect instrument to explore the inner reality which is exactly what we have been done all our lives when we think and when we understand the meaning and so on we are actually doing that in that Quantum reality we are not doing that in the brain
for - consciousness - takes place in quantum reality
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in your book one of the quotes was Free Will is the ultimate cause of reality
for - quote - free will is the ultimate cause of reality - Frederico Faggin
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fed Rico new book called irreducible
for - book - Irreducible - author - Federico Faggin - to - book Irreducible
to - book - Irreducible - https://hyp.is/0J8C4lo8Ee-WxX-r7RiEHw/www.collectiveinkbooks.com/essentia-books/our-books/irreducible-consciousness-life-computers-human-nature
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what you call CIP B which is the Consciousness information and physicality and how it links to opt which is operational probabilistic Theory
for - definition - Consciousness Information and Physicality (CIP) - definition - Operational Probabilistic Theory (OPT)
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it's evolution of this state of this Quantum state in hilber space which then will allow us to compute the probabilities of what you might measure in space and time it will not tell you generally what you will measure he only tells you the probability what you can measure and that's crazy in a sense right because classical objects you can actually described trajectory so that at any point in time you can tell position momentum and so on but not for Quantum Quantum system so so this fundamental difference will will see that is essential to describe why the Consciousness and Free Will must be must be Quantum phenomena
for - consciousness - quantum explanation depends on - difference between - quantum physics - and classical physics
consciousness - quantum explanation depends on - difference between - quantum physics - and classical physics - quantum state evolves in Hilbert space - enables computation of probabilities of what one measures in space-time - but doesn't tell you what you will measure - This difference is critical for describing consciousness as a quantum phenomena
Tags
- democracy of - the sacred
- hard problem of consciousness - Federico Faggin
- quantum physics - John Wheeler's theory - validating
- consciousness - primacy of in physics - quotations from famous scientists
- answer - how to experience nondual - how to experience non-separation and the authentic self - Federico Faggin
- definition - Consciousness Information and Physicality (CIP)
- epoche - kensho - satori - awakening experience - Federico Faggin
- Poverty mentality
- book - Irreducible - author - Federico Faggin
- consciousness - takes place in quantum reality
- Federico Faggin - high priority objective - find and implement ways to catalyze authentic awakening experiences for those who are ready
- AI - consciousness - not possible - Frederico Faggin
- definition - Operational Probabilistic Theory (OPT)
- Federico Faggin - high priority objective - find and implement ways to catalyze authentic awakening experiences in a short time - ie - one week
- to - book - Irreducible
- Federico Faggin - personal journey - profound awakening experience - reorientation of consciousness - from materialist - to idealist
- consciousness - quantum explanation depends on - difference between - quantum physics - and classical physics
- question - how to experience nondual - how to experience non-separation
- quote - free will is the ultimate cause of reality - Frederico Faggin
- CIP OPT integration - Federico Faggin
- quote - awakening experience - Federico Faggin
- comparison of death in - material vs idealist theories
- maya's illusion of separation
Annotators
URL
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The Best Ways to Remove Dried Hair Glue From Your Clothes
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What Is Mewing and How Do You Do It?
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learn.microsoft.com learn.microsoft.com#NoTDD1
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they know how to separate concerns and reduce coupling
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accessmedicina.mhmedical.com accessmedicina.mhmedical.com
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pleiotrópica,
una citocina previamente secretada puede generar diferentes efectos en una célula. Su receptor no está solamente en una célula
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preprints.jmir.org preprints.jmir.org
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Mark Eisenberg
This should be: Mark J. Eisenberg.
On the 3rd page of the pdf file, the affiliation should be: 1 Center for Clinical Epidemiology, Lady Davis Institute, Jewish General Hospital, Montreal, Quebec, Canada 2 Departments of Medicine and of Epidemiology, Biostatistics and Occupational Health, McGill University, Montreal, Quebec, Canada 4Department of Medicine, McGill University, Montreal, Quebec, Canada 5Division of Cardiology, Jewish General Hospital, McGill University, Montreal, Quebec, Canada
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Kristian Filiion
This should be: Kristian B. Filion. Also, on the 3rd page of the pdf file, the affiliation should be: 1 Center for Clinical Epidemiology, Lady Davis Institute, Jewish General Hospital, Montreal, Quebec, Canada 2 Departments of Medicine and of Epidemiology, Biostatistics and Occupational Health, McGill University, Montreal, Quebec, Canada
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Lynnette Lyzwinski
On the 3rd page of the pdf file, the affiliation should be: 1 Center for Clinical Epidemiology, Lady Davis Institute, Jewish General Hospital, Montreal, Quebec, Canada 2 Departments of Medicine and of Epidemiology, Biostatistics and Occupational Health, McGill University, Montreal, Quebec, Canada
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Meichen Dong;
Also, on the 3rd page of the pdf file, the affiliation should be: JMP Statistical Discovery, LLC, Cary, North Carolina, USA
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Russ Wolfinger
This should be: Russell D. Wolfinger. Also, on the 3rd page of the pdf file, the affiliation should be: JMP Statistical Discovery, LLC, Cary, North Carolina, USA
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ABSTRACT
On the third page of the pdf file, as indicated in the original paper, Mark J. Eisenberg should be the corresponding author. Also on the 3rd page, the authors affiliations are not correct.
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Lynnette Lyzwinski; Meichen Dong
These should be co-first authors
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www.theguardian.com www.theguardian.com
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en.wikipedia.org en.wikipedia.org
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SN 1181 belongs to a rare class of supernovas called Type Iax in which the thermonuclear flare-up could be the result of not one but two white dwarfs that have violently collided yet fail to detonate completely, leaving behind a “zombie star.”
So interesting!
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en.wikipedia.org en.wikipedia.org
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Heterodox economics
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en.wikipedia.org en.wikipedia.org
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International economics
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Local file Local file
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that whichmotivates the permeation of boundaries,
gullvers travels, heart of darkness
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hese twoenergie
complaint and eros
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en.wikipedia.org en.wikipedia.org
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Microeconomics
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en.wikipedia.org en.wikipedia.org
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Macroeconomics
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en.wikipedia.org en.wikipedia.org
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en.wikipedia.org en.wikipedia.org
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Applied economics
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www.derstandard.at www.derstandard.at
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www.biorxiv.org www.biorxiv.org
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Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
We thank the reviewers for going through our manuscript and providing valuable feedback. We are grateful to all 3 reviewers for describing our findings as important and valuable, well-designed and robust, and of value to the Parkinson's and Crohn's disease communities studying LRRK2. Below we detail a point-by-point response to the reviewers.
__Reviewer #1 (Evidence, reproducibility and clarity (Required)): __
The paper by Dikovskaya and collaborators investigated the activitiy and expression of LRRK2 in different subtypes of splenic and intestinal immune cells, taking advantage of a novel GFP-Lrrk2 knockin mouse. Interestingly, they found that T-cell-released IL-4 stimulates Lrrk2 expression in B cells. I have a few comments and suggestions for the authors. 1) Figure 1C. LRRK2 KO cells display residual Rab10 phosphorylation. Do the authors have any idea of which kinase other than LRRK2 could be involved in this phosphorylation?
As far as we are aware no other kinase is known to phosphorylate Rab10 at T73 in vivo. In vitro, recombinant Rab10 can be phosphorylated by MST3 at this site (Knebel A. et al, protocols.io https://dx.doi.org/10.17504/protocols.io.bvjxn4pn), but its relevance in vivo or in cells has not been shown. It is possible that the residual band recognised by anti-pT73 Rab10 ab in splenocytes is unspecific background, as it is mainly seen in LRRK2 KO spleen cells and not in other tissues. But to be certain that our assay assesses LRRK2-dependent Rab10 phosphorylation, we have always compared with the MLi-2 control.
2) Since there are no good antibodies for IF/IHC as pointed by the authors, the GFP-Lrrk2 mouse gives the opportunity to check endogenous LRRK2 localization, i.e. in cells untreated or treated with IL-4 or other cytokines. Also, does endogenous GFP-LRRK2 accumulate into filaments/puncta upon MLi2 inhibition? The relocalization into filaments of inhibited LRRK2 has been observed in overexpression but not under endogenous expression. This analysis would be interesting also in light of the observed side effect of type-I inhibitors.
We thank the reviewer for this suggestion. We will attempt a super-resolution microscopy using Airyscan with isolated B-cells treated with cytokine and/or LRRK2 inhibitor to address this question.
3) Figure 5. The authors need to label more clearly the graphs referring to wt mice versus GFP-Lrrk2 KI mice.
We have now labelled the panels referring to the WT mice only with "WT mice", to distinguish them from the other panels that incorporate data from both EGFP-Lrrk2 mice and their WT littermates used as a background.
They should also replace GFP-LRRK2 with GFP-Lrrk2 since they edited the endogenous murine gene.
Thank you, we have corrected it, and also the other mouse genotypes.
4) In the material and methods MLi-2 administration in mice is indicated at 60 mg/kg for 2 hr whereas in suppl. figure 5 the indicated dose is 30 mg/kg. Please correct with the actual dose used.
Thank you, we have corrected the mistake.
5) The discovery of IL-4 as a Lrrk2 activator in B cells is a very interesting and novel finding. The authors could take advantage of the GFP tag to investigate LRRK2 interactome upon IL-4 stimulation (optional). Also, is the signaling downstream of IL-4 attenuated in Lrrk2 KO cells?
We thank the reviewer for these interesting suggestions. The role of LRRK2 in IL-4 activated B-cells is currently under active research in the lab.
Reviewer #1 (Significance (Required)):
The manuscript is well designed and organized, and the experimental approaches are robust. These results are significant for the field as they add additional layers in the complex regulation and regulatory roles of LRRK2 in immunity, with implication for inflammatory disorders and Parkinson's disease.
We thank the reviewer for their positive comments and for recognising our efforts to provide some clarity to a complex field.
__Reviewer #2 (Evidence, reproducibility and clarity (Required)): __
The authors present a flow cytometry methodology to assess LRRK2 expression and pathway markers in mouse models and explore LRRK2 in splenic and intestinal immune cells. This is a highly valuable study given the emerging understanding that LRRK2 pathway activity in peripheral tissues may be of crucial importance to Parkinson's disease and Crohn's disease. P8 : the authors state that their results indicate 'that the effects of LRRK2-R1441C mutation and inflammation on LRRK2 activity represent two different parallel pathways'. This seems like an overinterpretation as pathway suggests the presence of additional partners in the pathway while R1441C is a LRRK2 intrinsic modification. The results can equally be explained by synergistic effects between both activation mechanisms (mutant and inflammation).
We agree with the reviewer, and have added this into the text. The sentence now reads "suggesting that the LRRK2-R1441C mutation and inflammation have different impacts on LRRK2 activity, either in parallel or in synergy."
Methods and experiment descriptions in results : the authors appear to use the terms anti-CD3 stimulation and CD3 stimulation interchangeably, although it is not always clear in the text that these are synonymous. This should be clarified.
We thank reviewer for pointing out this error on our part. We have made the necessary changes to always refer to the stimulation as anti-CD3.
One major observation in this paper is that LRRK2 is not detected in gut epithelial cells as previously has been reported. It would be useful to comment on any differences between the presented protocol and the previous reports, in particular relating to the antigen retrieval step. In order to reinforce the finding, it would be useful to include in situ hybridization data that could further strengthen the observations of which cellular subtypes express LRRK2 and which do not. Indeed, while the KO control shows that there is an unacceptable high non-specific staining, it does not prove absence of expression. Also, can any conclusions be made about expression of LRRK2 in neural cells of the gut? This important information on LRRK2 detection in gut should be mentioned in the abstract and highlighted in the discussion.
We thank the reviewer for pointing this out. In fact, we think the observation that LRRK2 is not detected in epithelial cells is so important that we have a separate manuscript exploring this point. Please see 1. Tasegian, A. et al.https://doi.org/10.1101/2024.03.07.582590 (2024). In this manuscript we have explored the expression of LRRK2 in human and murine intestinal epithelial cells using qPCR. Although we do not have in situ hybridization data, we believe that using both the EGFP-LRRK2 and the pRab10 flow cytometry, as well as qPCR and proteomics on selected cell types, corroborates our findings on the cell types that express LRRK2. We did not analyse LRRK2 expression in the neural cells of the gut, as the focus was on the immune cells, however we hope that others will use the tools developed here to explore this further.
The authors mention in the discussion that they 'show for the first time that eosinophils also express active LRRK2 at levels comparable to B-cells and DCs.' The relevance of this finding should be further developed. Why is this important?
We thank the reviewer for this point. We don't know how LRRK2 is important in these cells. However, as the role of LRRK2 in eosinophils and neutrophils has not yet been explored and both cell types play important roles in IBD, we think it is important to point out. We have now added a sentence to the discussion highlighting the importance of eosinophils in IBD. "Since eosinophils have recently been implicated as key player in intestinal defense and colitis(Gurtner et al, 2022), it will be interesting to evaluate LRRK2 functions in these cells."
In the isolation of lamina propria cells, what efforts were made to characterize the degree of purification of the lamina propria cells compared to cells of other gut wall layers such as epithelium, muscularis mucosa, or deeper layers? Please specify.
Isolation of lamina propria cells is a very well-established process (LeFrancois and Lycke, 'Isolation of Mouse Small Intestinal Intraepithelial Lymphocytes, Peyer's Patch, and Lamina Propria Cells.' Curr. Protocols in Immunology 2001), where we extensively wash off the epithelial layer before digesting the tissue for the LP. After the digestion the muscle and wall of the gut are still intact, so we do not get any contamination with other deeper layers. The subsets of cells we find in the LP are in line with isolations from other labs.
Minor comments Figure 5G, for the graphs indicating LRRK2 activity and LRRK2 phosphorylation, the specific measures should be specified in the graph titles to avoid any ambiguity (pT73-Rab10, pS935-LRRK2).
We have added the specifications to the new version of the figure.
Suppl figure 1 : please specify the figure label and abbreviation AF568 in the legend. Suppl figure 2 : please specify the figure label and abbreviation anti-rb in the legend
Thank you, we added the abbreviations to the legends. The Figure labels for both figures have been already included at the top of figure legends.
Reviewer #2 (Significance (Required)):
The authors present a flow cytometry methodology to assess LRRK2 expression and pathway markers in mouse models and explore LRRK2 in splenic and intestinal immune cells. This is a highly valuable study given the emerging understanding that LRRK2 pathway activity in peripheral tissues may be of crucial importance to Parkinson's disease and Crohn's disease.
We thank the reviewer for recognising the value of this study.
Reviewer #3
Evidence, reproducibility and clarity
The paper describes a set of experiments to analyse LRRK2 activity in tissues and despite it has very important findings and technical developments is largely descriptive. It does look like a collection of experiments more than a defined hypothesis and experiments to address that.
We thank the reviewer for recognising the importance of our findings and the technical developments. We agree that the paper's focus is to describe where LRRK2 is expressed in immune cells, and in which cells is it active or activated after inflammation in a hypothesis-free unbiased manner. We believe this is important data to share as a resource for the wider LRRK2 community and we will submit the manuscript as a Resource.
The flow cytometry assay of the first part is a great technical challenge and represents the establishment of a potentially very useful tool for the field. It would have been important to test other organs, either as controls or for example because of their relevance e.g. lungs. This first part is disconnected from the second part below.
We thank the reviewer for pointing out that the pRab10 assay would be useful to apply to other organs too. Since we are interested in the role of LRRK2 in IBD, we had focused on applying the pRab10 assay on intestinal tissue, with spleens also analysed as major lymphoid organ and a source of immune cells that can translocate to the gut in inflammation. We hope that the publication of this method would allow other researchers to analyse other tissues in the future.
The authors generated a new mouse KI mouse expressing EGFP-LRRK2 and show data the levels of LRRK2 expression are reduced in tissues at different degrees and established a flow cytometry assay to measure LRRK2 expression by monitoring the GFP signal. Interestingly they found that expression does not correlate with activity (as measured by phospho-Rabs). I suggest taking this part out as it breaks the flow of the paper. If data using this mouse is included, then microscopy should be included to complement the flow cytometry data. I understand the mice were used later with the anti-CD3 treatment, but it is very confusing that some experiments are done with EGFP-LRRK2 mice and others not. It does look in general like the mice do not behave as wild types and this is an important caveat. Without microscopy of the tissues or even cells (Figure 4) is hard to conclude much about these experiments.
We thank the reviewer for this point and would like to explain. It is true that in Suppl Figure 5, we show reduction of LRRK2 signal in the EGFP-Lrrk2-KI mice. However, based on immunoblotting, a significant reduction in EGFP-LRRK2 expression levels was seen only in the brain, but not in the tissues we analysed, that is the spleen and the intestine. Further, we have shown clearly using proteomics (Fig. 3D and 5E), that the GFP signal in immune cells correlates very well with the WT LRRK2 expression. Therefore, we think that the GFP signal in these mice reflects WT LRRK2 expression pattern. Further, despite the limitations of reduced kinase activity that we thoroughly describe, we think this model is very useful since no antibodies work to stain for LRRK2 in mice. We therefore respectfully disagree with this reviewer that the EGFP-LRRK2 data should be taken out, as it has proven to be an invaluable tool to measure and track changes in endogenous LRRK2 expression. Moreover, we think the fact that LRRK2 expression does not correlate with levels of activity, that is, LRRK2 is more active in some immune cells than in others, is a very important finding that evidences the cell-specific regulation of LRRK2 activity beyond its expression level.
We tried but failed to visualize the EGFP-LRRK2 signal using fluorescence microscopy in the tissue. This is most likely due to the low expression of LRRK2 (proteomics data suggests that even neutrophils express less than 9000 copies), confounded further by the high background autofluorescence in tissues, especially in the gut. We now explain the lack of tissue images from the EGFP-LRRK2 mice in the text. However, we can visualize the EGFP-LRRK2 in B cells, and we will provide these images in a revised version of the manuscript.
We have also added the following paragraph to the discussion:
"We complemented the pRab10 assay with the development of the EGFP-Lrrk2-KI reporter mouse. Although the reporter was initially designed as a fluorescent tracker for imaging LRRK2 localisation in cells and tissues, the low expression of LRRK2, combined with high and variable autofluorescence in tissues precluded its use for microscopy. Even in neutrophils, which express highest level of LRRK2 among immune cells, there are less than 9000 copies of LRRK2 per cell (Sollberger et al, 2024), making it difficult to identify localization. However, the EGFP signal was sufficient for flow cytometry-based measurements, where background autofluorescence of each cell type was taken into account and subtracted."
Then the authors show that LRRK2 expression and activity is different in different cell types and depends on inflammation. The anti-CD3 strategy to induce inflammation is very different from physiological inflammation such as sepsis and LPS stimulation, so experiments with other stimuli could be important here to contribute to the message of inflammatory trigger of LRRK2 activation and decoupling of cell type.
We thank the reviewer for this suggestion. We used the anti-CD3 model as it also causes intestinal inflammation, and mimics T-cell cytokine storms that happens in many diseases. However, for the revisions we will also test another model of inflammation as suggested, such as LPS stimulation, to measure how inflammation affects LRRK2 expression and activity.
The IL-4 data is intriguing but too preliminary. The lack of strong effect of IFN-gamma is expected as the promoter of LRRK2 in mice and humans is different and human cells responds much better with regards to LRRK2 expression after IFN-gamma stimulation.
We are confused by what the reviewer means by saying the IL-4 data is preliminary. We have shown by flow cytometry, immunoblotting, qPCR and proteomics that IL-4 induced LRRK2 expression in B-cells. So we are uncertain as to how else this can be shown. As to the effect of IFNγ on LRRK2 expression, it may indeed be that human cells respond better than murine cells. Importantly, the IL-4 ability to induce LRRK2 in B-cells is a novel and important finding, regardless of the effects of IFNγ.
Reviewer #3 (Significance (Required))
The paper describes a set of experiments to analyse LRRK2 activity in tissues and despite it has very important findings and technical developments is largely descriptive. It does look like a collection of experiments more than a defined hypothesis and experiments to address that.
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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #3
Evidence, reproducibility and clarity
The authors present a flow cytometry methodology to asses LRRK2 epxression and pathway markers in mouse models and explore LRRK2 in splenic and intestinal immune cells. This is a highly valuable study given the emerging understanding that LRRK2 pathway activity in peripheral tissues may be of crucial importance to Parkinson's disease and Crohn's disease.
P8 : the authors state that their results indicate 'that the effects of LRRK2-R1441C mutation and inflammation on LRRK2 activity represent two different parallel pathways'. This seems like an overinterpretation as pathway suggests the presence of additional partners in the pathway while R1441C is a LRRK2 intrinsic modification. The results can equally be explained by synergistic effects between both activation mechanisms (mutant and inflammation).
Methods and experiment descriptions in results : the authors appear to use the terms anti-CD3 stimulation and CD3 stimulation interchangeably, although it is not always clear in the text that these are synonymous. This should be clarified.
One major observation in this paper is that LRRK2 is not detected in gut epithelial cells as previously has been reported. It would be useful to comment on any differences between the presented protocol and the previous reports, in particular relating to the antigen retrieval step. In order to reinforce the finding, it would be useful to include in situ hybridization data that could further strengthen the observations of which cellular subtypes express LRRK2 and which do not. Indeed, while the KO control shows that there is an unacceptable high non-specific staining, it does not prove absence of expression. Also, can any conclusions be made about expression of LRRK2 in neural cells of the gut? This important information on LRRK2 detection in gut should be mentioned in the abstract and highlighted in the discussion. The authors mention in the discussion that they 'show for the first time that eosinophils also express active LRRK2 at levels comparable to B-cells and DCs.' The relevance of this finding should be further developed. Why is this important ?
In the isolation of lamina propria cells, what efforts were made to characterize the degree of purification of the lamina propria cells compared to cells of other gut wall layers such as epithelium, muscularis mucosa, or deeper layers? Please specify.
Minor comments
Figure 5G, for the graphs indicating LRRK2 activity and LRRK2 phosphorylation, the specific measures should be specified in the graph titles to avoid any ambiguity (pT73-Rab10, pS935-LRRK2).
Suppl figure 1 : please specify the figure label and abbreviation AF568 in the legend.
Suppl figure 2 : please specify the figure label and abbreviation anti-rb in the legend
Significance
The authors present a flow cytometry methodology to asses LRRK2 epxression and pathway markers in mouse models and explore LRRK2 in splenic and intestinal immune cells. This is a highly valuable study given the emerging understanding that LRRK2 pathway activity in peripheral tissues may be of crucial importance to Parkinson's disease and Crohn's disease.
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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
The paper by Dikovskaya and collaborators investigated the activitiy and expression of LRRK2 in different subtypes of splenic and intestinal immune cells, taking advantage of a novel GFP-Lrrk2 knockin mouse. Interestingly, they found that T-cell-released IL-4 stimulates Lrrk2 expression in B cells.
I have a few comments and suggestions for the authors.
- Figure 1C. LRRK2 KO cells display residual Rab10 phosphorylation. Do the authors have any idea of which kinase other than LRRK2 could be involved in this phosphorylation?
- Since there are no good antibodies for IF/IHC as pointed by the authors, the GFP-Lrrk2 mouse gives the opportunity to check endogenous LRRK2 localization, i.e. in cells untreated or treated with IL-4 or other cytokines. Also, does endogenous GFP-LRRK2 accumulate into filaments/puncta upon MLi2 inhibition? The relocalizaiton into filaments of inhibited LRRK2 has been observed in overexpression but not under endogenous expression. This analysis would be interesting also in light of the observed side effect of type-I inhibitors.
- Figure 5. The authors need to label more clearly the graphs referring to wt mice versus GFP-Lrrk2 KI mice. They should also replace GFP-LRRK2 with GFP-Lrrk2 since they edited the endogenous murine gene.
- In the material and methods MLi-2 administration in mice is indicated at 60 mg/kg for 2 hr whereas in suppl. figure 5 the indicated dose is 30 mg/kg. Please correct with the actual dose used.
- The discovery of IL-4 as a Lrrk2 activator in B cells is a very interesting and novel finding. The authors could take advantage of the GFP tag to investigate LRRK2 interactome upon IL-4 stimulation (optional). Also, is the signaling downstream of IL-4 attenuated in Lrrk2 KO cells?
Significance
The manuscript is well designed and organized, and the experimental approaches are robust. These results are significant for the field as they add additional layers in the complex regulation and regulatory roles of LRRK2 in immunity, with implication for inflammatory disorders and Parkinson's disease.
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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
The paper describes a set of experiments to analyse LRRK2 activity in tissues and despite it has very important findings and technical developments is largely descriptive. It does look like a collection of experiments more than a defined hypothesis and experiments to address that.
The flow cytometry assay of the first part is a great technical challenge and represents the establishment of a potentially very useful tool for the field. It would have been important to test other organs, either as controls or for example because of their relevance e.g. lungs. This first part is disconnected from the second part below.
The authors generated a new mouse KI mouse expressing EGFP-LRRK2 and show data the levels of LRRK2 expression are reduced in tissues at different degrees and established a flow cytometry assay to measure LRRK2 expression by monitoring the GFP sugnal. Interestengly they found that expression does not correlate with activity (as measured by phospho-Rabs). I suggest taking this part out as it breaks the flow of the paper. If data using this mouse is included, then microscopy should be included to complement the flow cytometry data. I understand the mice were used later with the anti-CD3 treatment, but it is very confusing that some experiments are done with EGFP-LRRK2 mice and others not. It does look in general like the mice do not behave as wild types and this is an important caveat. Without microscopy of the tissues or even cells (Figure 4) is hard to conclude much about these experiments.
Then the authors show that LRRK2 expression and activity is different in different cell types and depends on inflammation. The anti-CD3 strategy to induce inflammation is very different from physiological inflammation such as sepsis and LPS stimulation, so experiments with other stimuli could be important here to contribute to the message of inflammatory trigger of LRRK2 activation and decoupling of cell type.
The IL-4 data is intriguing but too preliminary. The lack of strong effect of IFN-gamma is expected as the promoter of LRRK2 in mice and humans is different and human cells responds much better with regards to LRRK2 expression after IFN-gamma stimulation.
Significance
The paper describes a set of experiments to analyse LRRK2 activity in tissues and despite it has very important findings and technical developments is largely descriptive. It does look like a collection of experiments more than a defined hypothesis and experiments to address that.
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cdn.cdp.net cdn.cdp.net
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The taxonomy is where the rules and data definitions are organised. It is comprisedof a set of elements (i.e., Key Performance Indicatorsand narratives) and all the presentation,calculation and standard logic rules that are in effect. Once created, the XBRL taxonomy is made public as an open sourcefileon the internet. Then, for a specific firm, software can be used to create an XBRL instance (the report itself), containing the specific facts and figures for a certain period. The XBRL instance can be checked against the taxonomy by all parties (reporting entity, a regulator, or even the public) in order toguarantee its data qualityand reliability, as the taxonomy contains data quality checks that any XBRL engine canvalidate
This is actually a handy description
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Africans have arrived with divergent views of the borough and its people. Some were exposed to American movies, which depicted Harlem as a neighborhood rife with crime and chaos. Others consumed Black popular culture or read the words of civil rights leaders. In other words, while some have already internalized negative images of Harlem before they arrived, many others consider it their very own “Black Mecca.” They are attracted not only to its glorious past but also to its Black soul, its blues impulse. Because they are emigrating from African countries, they find that Black neighborhoods imbued with this sensibility can feel like home. Of course, the reality is often quite different, especially when class conflicts and cultural misfires go unresolved.
diaspora wars almost. you view african americans as one way but you come and it is different
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and moralists (who were opposed to nuclear in principle)
What is the moral argument against nuclear (power, not bombs)?
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for - from - interview - Federico Faggin
from - interview - Federico Faggin - https://hyp.is/Bmcmblo_Ee-cuC8QnSOAYw/www.youtube.com/watch?v=MSn4t6fP_dc
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imary role of perceptual processes was to guide action
i.e., informing us of where we are in space and time so as to inform future action?
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alia
qualia!!!
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sellercentral.amazon.fr sellercentral.amazon.frAmazon1
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Elle démarre dans notre Van aménagé. Nous sommes Clem et Nico, un duo d'aventuriers ingénieux, adeptes de la Van life, du camping, et du backpack travel. Notre objectif : vous permettre de profiter pleinement de vos aventures nomades et sédentaires grâce à notre gamme de voyage accessoires.
Changer le texte par …
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As a result we reach a quantum-information-based panpsychism, with classical physics supervening on quantum physics, quantum physics supervening on quantum information, and quantum information supervening on consciousness.
for - quantum-information-based-panpsychism - consciousness - relationship - quantum information - to consciousness
consciousness - relationship - quantum information - to consciousness - classical physics supervenes on quantum physics - quantum physics supervenes on quantum information - quantum information supervenes on consciousness
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We also see how the same purity of state and evolution allow one to solve the well-known combination problem of panpsychism.
for - follow up - combination problem of panpsychism
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How to Tell if You Have Started Puberty (for Girls)window.WH=window.WH||{};window.WH.c=function(){function C(){var a=D().split("x")[0];return"undefined"!=typeof g.largeScreenMinWidth&&a&&parseInt(a,10)<g.largeScreenMinWidth?1:0}function p(){var a=E;if(h){var b=+new Date-h;0<b&&(a+=b)}return a}function F(a){if("undefined"!==typeof a&&"undefined"!==typeof a.target&&"undefined"!==typeof a.target.getAttribute&&(a=a.target.getAttribute("id"),"undefined"!==typeof a&&0!==a.indexOf("whvid-player")))return;G||(G=!0,k&&6>=k||(a=q({d:x?C()?"vm":"vw":"pv",m:g.pageName+" btraw "+p()/1E3,b:"6"}),delete a.dl,r("exit",a,!0)))}function H(){h&&(E+=+new Date-h,h=!1)}function I(){h||(h=+new Date)}function J(){var a=document.querySelectorAll("#intro, .section.steps, #quick_summary_section");if(a){var b=1E6,e=0;Array.prototype.forEach.call(a,function(a){var c=a;for(var d=0;c;)d+=c.offsetTop-c.clientTop,c=c.offsetParent;c=d;a=a.offsetHeight||a.clientHeight;b=Math.min(c,b);e=Math.max(c+a,e)});n=b;y=e}}function U(){var a=!1;try{var b=Object.defineProperty({}, "passive",{get:function(){a=!0}});window.addEventListener("testPassive",null,b);window.removeEventListener("testPassive",null,b)}catch(e){}window.addEventListener("scroll",function(){f++;if(-1!=t){var a=+new Date-u;a>=t+250&&(t=a,J())}var b=p();a=b-K;K=b;if(!(0>=a)){var c=window.scrollY||window.pageYOffset;b=c+(window.innerHeight||document.documentElement.clientHeight);if(!(c>y||b<n)){var d=y-n;if(!(0>=d))for(c=Math.floor(128*(1*c-n)/d),0>c&&(c=0),b=Math.ceil(128*(1*b-n)/d),127<b&&(b=127),d=c;d<=b;)"undefined"===typeof v[d]&&(v[d]=0),v[d]+=a,d++}}});window.addEventListener("resize",function(){f++});window.addEventListener("click",function(){f++});b=a?{passive:!0}:!1;window.addEventListener("touchstart",function(){f++},b);window.addEventListener("touchend",function(){f++});window.addEventListener("touchcancel",function(){f++});window.addEventListener("touchmove",function(){f++},b);document.addEventListener("keydown",function(){f++});document.addEventListener("keyup",function(){f++}); 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How to Know when You Are Going to Start Puberty (Girls)
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So have little way to mediate the #closedweb problem of the groups who “succeed” in a capitalist being the worst equipped to solve the problems that the system creates.
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eLife assessment
This is a mechanistic study showing the effect of combining inhibition of autophagy (through ULK1/2) and KRAS (using sotorasib) on KRAS mutant NSCLC making the study valuable to cancer biologists and more broadly in a clinical setting. The evidence generated by GEM mouse models and cell lines is solid but could be further strengthened by increasing the mouse cohort size. This study holds translational relevance beyond NSCLC to other indications that carry KRAS mutations.
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Reviewer #2 (Public Review):
Summary:
In this manuscript, Ghazi et reported that inhibition of KRASG12C signaling increases autophagy in KRASG12C expressing lung cancer cells. Moreover, the combination of DCC 3116, a selective ULK1/2 inhibitor, plus sotorasib displays cooperative/synergistic suppression of human KRASG12C driven lung cancer cell proliferation in vitro and tumor growth in vivo. Additionally, in genetically engineered mouse models of KRASG12C driven NSCLC, inhibition of either KRASG12C or ULK1/2 decreases tumor burden and increases mouse survival. Additionally, this study found that LKB1 deficiency diminishes the sensitivity of KRASG12C/LKB1Null-driven lung cancer to the combination treatment, perhaps through the emergence of mixed adeno/squamous cell carcinomas and mucinous adenocarcinomas.
Strengths:
Both human cancer cells and mouse models were employed in this study to illustrate that inhibiting ULK1/2 could enhance the responsiveness of KRASG12C lung cancer to sotorasib. This research holds translational importance.
Weaknesses:
The revised manuscript has addressed most of my previous concerns. However, I still have one issue: the sample size (n) for the GEMM study in Figures 4E and 4F is too small, despite the authors' explanation. The data do not support the conclusion due to the lack of significant difference in tumor burden. Additionally, the significance labels in Figure 4E are not clearly explained.
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Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public Review):
Summary:
Given that KRAS inhibition approaches are a relatively new innovation and that resistance is now being observed to such therapies in patients with NSCLC, investigation of combination therapies is valuable. The manuscript furthers our understanding of combination therapy for KRAS mutant non-small cell lung cancer by providing evidence that combined inhibition of ULK1/2 (and therefore autophagy) and KRAS can inhibit KRAS-mutant lung cancer growth. The manuscript will be of interest to the lung cancer community but also to researchers in other cancer types where KRAS inhibition is relevant.
Strengths:
The manuscript combines cell line, cell line-derived xenograft, and genetically-engineered mouse model data to provide solid evidence for the proposed combination therapy. The manuscript is well written, and experiments are broadly well performed and presented.
We thank Reviewer #1 (R1) for the generally favorable review of our manuscript, and also for the more detailed critique that identifies potential weaknesses in the research, which we address on a point-by-point basis below.
Weaknesses:
With 3-4 mice per group in many experiments, experimental power is a concern and some comparisons (e.g. mono vs combination therapy) seem to be underpowered to detect a difference. Both male and female mice are used in experiments which may increase variability.
We thank R1 for pointing out concerns regarding statistical power in our various mouse models of NSCLC experiments, and agree that more mice per group would certainly increase statistical power. However, there are certain logistical considerations that impact the generation of cohorts of experimental KrasLSL-G12C mice. Because mice homozygous for the KrasLSL-G12C allele display embryonic lethality, we are required to generate experimental mice by crossing heterozygous male and female KrasLSL-G12C mice. Although 66% of the progeny of such crosses are predicted to be KrasLSL-G12C/+, experience tells us that we only obtain ~40-50% heterozygous KrasLSL-G12C/+ mice with litter sizes around 6-8 mice from such crosses. Therefore, there are usually only about 4 heterozygous KrasLSL-G12C mice per litter, which presents a substantial challenge in generating larger cohorts of age-matched mice suitable for experiments, especially under conditions where we wish to euthanize mice at multiple time points for analysis. For the GEM model experiments, Figure 3B is the only experiment that has n=3. All other experiments contain 4-6 mice per experimental condition. We rationalized using both male and female mice because both human males and females have high lung cancer rates.
Reviewer #2 (Public Review):
Summary:
In this manuscript, Ghazi et reported that inhibition of KRASG12C signaling increases autophagy in KRASG12C-expressing lung cancer cells. Moreover, the combination of DCC 3116, a selective ULK1/2 inhibitor, plus sotorasib displays cooperative/synergistic suppression of human KRASG12C-driven lung cancer cell proliferation in vitro and tumor growth in vivo. Additionally, in genetically engineered mouse models of KRASG12C-driven NSCLC, inhibition of either KRASG12C or ULK1/2 decreases tumor burden and increases mouse survival. Additionally, this study found that LKB1 deficiency diminishes the sensitivity of KRASG12C/LKB1Null-driven lung cancer to the combination treatment, perhaps through the emergence of mixed adeno/squamous cell carcinomas and mucinous adenocarcinomas.
Strengths:
Both human cancer cells and mouse models were employed in this study to illustrate that inhibiting ULK1/2 could enhance the responsiveness of KRASG12C lung cancer to sotorasib. This research holds translational importance.
We thank Reviewer #2 (R2) for the generally favorable review of our manuscript, and also for the more detailed critique that identifies potential weaknesses in the research, which we address on a point-by-point basis below.
Weaknesses:
Additional validation of certain data is necessary.
(1) mCherry-EGFP-LC3 reporter was used to assess autophagy flux in Figure 1A. Please explain how autophagy status (high, medium, and low) was defined. It's also suggested to show WB of LC3 processing in different treatments as in Figure 1A at 48 hours.
We thank the reviewer for this comment and agree that a more thorough description of how autophagy status is assessed using the Fluorescent Autophagy Reporter (FAR) would benefit the readers of our manuscript. Cells engineered to express the FAR are analyzed by flow cytometry in which we defined autophagy status by gating viable (based Sytox Blue staining), DMSO-treated control cells into three bins based on the ratio of EGFP:mCherry fluorescence. We gate all live cells into the 33% highest EGFP-positive cells (autophagy low) and the 33% highest mCherry-positive cells (autophagy high), and therefore, the proportion in the middle is also approximately 33% and considered the medium autophagy status. Again, these gates are based entirely on the DMSO-treated control cells, and all other treatments within the experiment are compared to settings on these gates. In response to a specific manipulation (sotorasib, trametinib, DCC-3116 etc) we assess how the specific treatment changes the percentages of cells in each of the pre-specified gates to assess increased autophagy (decreased EGFP:mCherry ratio) or decreased autophagy (increased increased EGFP:mCherry ratio).
Although LC3 processing and/or the expression of p62SQSTM1 are used by others as markers of autophagy, there is much debate in the literature as to how reliable immunoblotting analysis of LC3 processing or p62SQSTM1 expression are as measures of autophagy. Certainly, in our hands, we find that the Fluorescent Autophagy Reporter is a much more sensitive measure of changes in autophagy in various different cancer cell lines as we have described in previous papers (Kinsey et al., PMID: 30833748, Truong et al., PMID: 32933997 and Silvis & Silva et al., PMID: 36719686). Furthermore, in the omnibus publication that describes techniques for measuring autophagy (Klionsky et al., PMID: 33634751) the use of the FAR (or similarly configured reporters) is regarded as the gold standard for measuring autophagy status in cells. We have amended the Materials & Methods section of our manuscript to better describe the use of the FAR in measuring autophagy.
(2) For Figures 1J, K, and L, please provide immunohistochemistry (IHC) images demonstrating RAS downstream signaling blockade by sotorasib and autophagy blockade by DCC 3116 in tumors.
We thank the reviewer for the comment and have probed the tumors from the xenograft experiments in Figures 1J, K, and L for pERK1/2 and p62SQSTM1 to determine the biochemical activity of sotorasib or DCC-3116, respectively and have provided representative images below. We observed the expected decrease in pERK and p62 signal after sotorasib treatment in all three xenografted cell lines. We did observe the expected accumulation of p62 in the DCC-3116 treated tumors from the NCI-H2122 and NCI-H358 cell lines. There appears to be no difference between the vehicle and DCC-3116 treated tumors in the NCI-H358 cell line-derived tumors as detected by IHC.
Author response image 1.
(3) Given that both DCC 3116 and ULK1K46N exhibit the ability to inhibit autophagy and synergize with sotorasib in inhibiting cell proliferation, in addition to demonstrating decreased levels of pATG13 via ELISA assay, please include Western blot analyses of LC3 or p62 to confirm the blockade of autophagy by DCC 3116 and ULK1K46N in Figure 1 & Figure 2.
We appreciate the reviewer's comment and have performed an immunoblot analysis of cells treated with DCC-3116 or expressing ULK1K46N and probed for p62SQSTM1 and LC3 expression. We did observe the expected accumulation of p62 SQSTM1 in NCI-H2122 (ULK1K46N) cells treated with 1ug/ml doxycycline to induce expression of ULK1K46N compared to DMSO treatment. Additionally, we treated the human cell lines from Figure 1 with sotorasib and/or DCC-3116 and tested for p62SQSTM1 expression after 48 hours of treatment. In the human cell lines NCI-H2122 and NCI-H358, there was a decrease in the p62 signal with increasing doses of sotorasib, as expected. There was no detectable change in p62 levels in the Calu-1 cells by immunoblot. For LC3-I/LC3-II, there was only one detectable band in the NCI-H2122 cells, which makes it difficult to interpret the results and further emphasizes why we use the fluorescent autophagy reporter which is more sensitive than immunoblotting. There is no detectable change in LC3-I/LC3-II in the Calu-1 cells treated with increasing doses of sotorasib, but the expected decrease in LC3-I is observed with sotorasib treatment in the NCI-H358 cells.
Author response image 2.
(4) Since adenocarcinomas, adenosquamous carcinomas (ASC), and mucinous adenocarcinomas were detected in KL lung tumors, please conduct immunohistochemistry (IHC) to detect these tumors, including markers such as p63, SOX2, Katrine 5.
We have included IHC analysis of the adenosquamous carcinomas for the markers p63, SOX2, and Keratin 5 from the KL mouse in Figure 3 and the ASC tumors in Supplemental Figure 4, and thank the reviewer for this excellent suggestion. The straining for these markers is below. Of note, we tried two different SOX2 antibodies (cell signaling technologies #14962 and cell signaling technologies # 3728) and could not detect any staining in any section.
Author response image 3.
(5) Please provide the sample size (n) for each treatment group in the survival study (Figure 4E). It appears that all mice were sacrificed for tumor burden analysis in Figure 4F. However, there doesn't seem to be a significant difference among the treatment groups in Figure 4F, which contrasts with the survival analysis in Figure 4E. It is suggested to increase the sample size in each treatment group to reduce variation.
We have updated Figure 4E to indicate sample size for each treatment group and thank the reviewer for this suggestion. Any mice that remained on study through the entire 8-week treatment regimen were sacrificed after the last day of treatment (Day 56). Figure 4F indicates analysis of total tumor burden in all mice that remained on treatment for the full 8 weeks and mice that reached euthanasia criteria before the end of the 8-week treatment. Therefore, it is important to note that the mice in Figure 4F were not all euthanized on the same day. There is no statistically significant difference between the 3 treatment groups (sotorasib, DCC-3116, combination). This may be due to a lower sample size as well as ending the treatment at 8 weeks as opposed to continuing the treatment for a longer period of time. Although we agree that increasing the sample size would benefit the study, due to how long the GEMM model experiments take (12-16 weeks of breeding, 6 weeks for the mice to reach adulthood, 10 weeks of tumor formation post-initiation, 8 weeks of treatment= ~40 weeks) we would respectfully submit that the analysis of additional mice is outside the scope of the current revised manuscript.
(6) In KP mice (Figure 5), it seems that a single treatment alone is sufficient to inhibit established KP lung tumor growth. Combination treatment does not further enhance anti-tumor efficacy. Therefore, this result doesn't support the conclusion generated from human cancer cell lines. Please discuss.
We thank the reviewer for this observation. Indeed, KP lung tumors were sensitive to single agent DCC-3116 treatment, which is reflected in the tumor burden analysis. This was somewhat surprising to us as we have not previously detected much anti-tumor activity using 4-amino-quinoloines (chloroquine or hydroxychloroquine) or other autophagy inhibitors. It should be noted however that the KRASG12C/TP53R175H NSCLC model has a very low tumor burden overall (~4% in vehicle-treated mice). Additionally, our microCT imager cannot detect AAH and small tumors at the settings/resolution used. Therefore, we were limited in our ability to detect small tumors or hyperplasia by microCT imaging. Although there was a decrease in overall tumor burden with single agent DCC-3116 treatment, we could not demonstrate using microCT imaging that KRASG12C/TP53R175H lung tumors were actually regressing with single agent DCC-3116 treatment. The larger tumors that were detected appeared to show a cytostatic effect (i.e. no or slow growth) with DCC-3116 monotherapy. This may reflect our inability to detect regression of AAH or small tumors with the microCT. In all human cell lines tested, the only cell line that responded to single agent DCC-3116 treatment was NCI-H358 cells, which do have a complete heterozygous loss of the TRP53 gene and lack TP53 protein. However, other cells that also have a loss of expression of TP53 expression (Calu-1) are insensitive to single-agent DCC-3116 treatment. Due to the low mutational burden of the KP mouse model compared to human NSCLC cell lines driven by mutationally-activated KRASG12C and the loss of TP53 function, it is difficult to directly compare GEM models to the human cell line models. Most of the human cell lines have alterations in other genes that are not altered in the KP mouse model which could affect the sensitivity of treatment.
Recommendations for the authors:
Reviewer #1 (Recommendations For The Authors):
Minor comments:
(1) Figure legends are currently not adequate - information about the number and nature of replicates, stats, and definitions of the labelling used for stats should be added throughout. In Figure 5B, only two lines of four are labelled with * or ns.
We thank the reviewer for this comment and have included more details in the figure legends that describe replicates, statistical analysis and definitions of labeling. We also note that the methods section has a detailed description of the statistical analysis used.
(2) What statistical test is performed on Figure 5E to get a p < 0.05 between the vehicle and DCC group?
We performed a one-way ANOVA for all statistical analyses with more than 2 experiential groups. We thank the reviewer for pointing out this typo. These data points (vehicle vs. DCC-3116) are not statistically significant, which has been revised in the figure.
(3) The manuscript figures would be improved by the use of a colourblind-friendly palette.
We have previously published multiple manuscripts using this color scheme for the fluorescent autophagy reporter experiments and chose to use red and green as the reporter uses EGFP and mCherry. We wanted to keep this color scheme consistent across our publications and would prefer not to change the colors. However, we agree with the reviewer that the data should be accessible to all people and, therefore, have updated these graphs to include slashes over the red color to ease in telling the differences between the red and green colors. Thank you to the reviewer for this excellent suggestion.
(4) The manuscript should be fully checked for mouse (sentence case) and human (caps) gene (italics) and protein (non-italics).
In this manuscript we are using the nomenclatures approved by the HUGO Gene Nomenclature Committee (https://en.wikipedia.org/wiki/HUGO_Gene_Nomenclature_Committee) in which:
Human genes are written as KRAS, TP53 etc i.e. ITALICIZED CAPS
Mouse genes are written as Kras, Trp53 etc: i.e. Italicized and sentence case
Human and mouse proteins are written as KRAS, TP53 etc: i.e. NON-ITALICIZED CAPS
In response to the reviewer’s suggestion, we have gone through the manuscript to check for this and make any appropriate changes. Of note, we intentionally refer to the mouse protein changes as KRASG12C/LKB1null or KRASG12C/TP53R172H (capitalized), as this references the protein change and not the nucleotide change that occurs in the gene.
(5) Adenosquamous is the correct term for the disease. In parts, it's referred to as adeno/squamous or adeno-squamous. The abbreviation ADC is also defined many times.
Thank you to the reviewer for this comment. We have corrected the manuscript text to only use adenosquamous and only define ADC in the first instance.
(6) Line 434 - "as previously described" but no reference.
Typos:
(1) Line 117 – either
(2) Line 314 – synergistic
(3) Line 317 – therefore
(4) Line 502 – medium
We thank the reviewer for pointing out these typos and have modified the text appropriately.
Reviewer #2 (Recommendations For The Authors):
(1) The statement on Page 4, Lines 119-120, lacks clarity: 'Furthermore, LKB1 silencing diminishes the sensitivity of KRASG12C/LKB1Null-driven lung cancer perhaps through the emergence of mixed adeno/squamous cell carcinomas and mucinous adenocarcinomas. It is unclear whether this refers to the sensitivity to the combination treatment or to the KRASc inhibitor alone.
We thank the reviewer for this comment and agree that the statement lacks clarity. The intent of this statement was to refer to both single agent sotorasib treatment as well as the combination with DCC-3116.
(2) Page 5 Line 147 "KRASG12X ". Please correct this typo.
We thank the reviewer for this comment, but this is not a typo. We intended for this line to state KRASG12X to refer to cell lines with any KRASG12 alteration, e.g KRASG12D, KRASG12C, KRASG12S, KRASG12R etc.
(3) The color of the dots in Figure 5B labeling does not match the dots in the graph.
For all bar graphs in the manuscript, the dots representing individual mice are black, and the bar itself is color-coded based on treatment type. The dots in Figure 5B follow this pattern and are intended to be this way.
(4) Figure 5C depicts lung weight rather than tumor growth, contrary to the text description "regression of pre-existing lung tumors was detected by microCT scanning (Figure 5C, Figure S5)".
Figure 5C does not depict lung weight but the percent body weight change in treated mice, described in the figure legend. We thank the reviewer for pointing this out because we referenced the wrong panel in the text. The figures referenced should be Figure 5B, Figure S5. We have corrected this in the text.
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Reviewer #1 (Public Review):
Summary:
The authors profile gene expression, chromatin accessibility and chromosomal architecture (by Hi-C) in activated CD4 T cells and use this information to link non-coding variants associated with autoimmune diseases with putative target genes. They find over a 1000 genes physically linked with autoimmune disease loci in these cells, many of which are upregulated upon T cell activation. Focusing on IL2, they dissect the regulatory architecture of this locus, including the allelic effects of GWAS variants. They also intersect their variant-to-gene lists with data from CRISPR screens for genes involved in CD4 T cell activation and expression of inflammatory genes, finding enrichments for regulators. Finally, they showed that pharmacological inhibition of some of these genes impacts T cell activation.
This is a solid study that follows a well-established canvas for variant-to-gene prioritisation using 3D genomics, applying it to activated T cells. The authors go some way in validating the lists of candidate genes, as well as explore the regulatory architecture of a candidate GWAS locus. Jointly with data from previous studies performing variant-to-gene assignment in activated CD4 T cells (and other immune cells), this work provides a useful additional resource for interpreting autoimmune disease-associated genetic variation.
Autoimmune disease variants were already linked with genes in CD28-stimulated CD4 T cells using chromosome conformation capture, specifically Promoter CHi-C and the COGS pipeline (Javierre et al., Cell 2016; Burren et al., Genome Biol 2017; Yang et al., Nat Comms 2020). The authors cite these papers and present a comparative analysis of their variant-to-gene assignments (in addition to scRNA-seq eQTL-based assignments). Furthermore, they find that the Burren analysis yields a higher enrichment for gold standard genes.
I thank the authors for their revisions in response to my initial review. The revised version now includes a more comprehensive comparative analysis of different datasets and V2G approaches and discusses the potential sources of differences in the results. Most significantly, the authors have now included an interesting comparison of their methodology with the popular ABC technique and outlined the key limitations of ABC relative to their method and other (Capture) Hi-C-based V2G approaches.
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www.researchsquare.com www.researchsquare.com
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eLife assessment
This is a useful manuscript describing the competitive binding between Parkin domains to define the importance of dimerization in the mechanism of Parkin regulation and catalytic activity. The evidence supporting the importance of Parkin dimerization for an 'in trans' model of Parkin activity described in this manuscript is solid, but lacks more stringent and biochemical characterization of competitive binding that could provide more direct evidence to support the author's conclusions. This work will be of interest to those focused on defining the molecular mechanisms involved in ubiquitin ligase interactions, PINK-Parkin-mediated mitophagy, and mitochondrial organellar quality control.
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Reviewer #1 (Public Review):
Summary:
The authors used structural and biophysical methods to provide insight into Parkin regulation. The breadth of data supporting their findings was impressive and generally well-orchestrated.
Strengths:
(1) They have done a better job explaining the rationale for their experiments thought-out.
(2) The use of molecular scissors in their construct represents a creative approach to examine inter-domain interactions. Appropriate controls were included.
(3) From my assessment, the experiments are well-conceived and executed.
(4) The authors do a better job of highlighting the question being addressed experimentally.
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Reviewer #2 (Public Review):
In the revised manuscript, the authors tried to address some of my comments from the previous round of review. Notably, they have performed some additional ITC experiments where protein precipitation is not an issue to probe interactions between PARKIN and different domains. In addition, they have toned down some of the language in the text to better reflect their data and results. However, I still feel that the manuscript lacks some key answers regarding the relative interactions between p-PARKIN and different domains, as discussed in my previous review. A deeper dive into the underlying biophysical and biochemical features that drive these interactions is important to fully understand the importance of their work. However, this manuscript does provide some interesting potential insights into the mechanisms of PARKIN activation that could be useful for the field moving forward.
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Reviewer #3 (Public Review):
Summary:
In their manuscript, Lenka et al present data that could suggest an "in trans" model of Parkin ubiquitination activity. Parkin is an intensely studied E3 ligase implicated in mitophagy, whereby missense mutations to the PARK2 gene are known to cause autosomal recessive juvenile parkinsonism. From a mechanistic point of view, Parkin is extremely complex. Its activity is tightly controlled by several modes of auto-inhibition that must be released by queues of mitochondrial damage. While the general overview of Parkin activation has been mapped out in recent years, several details have remained murky. In particular, whether Parkin dimerizes as part of its feed-forward signaling mechanism, and whether said dimerization can facilitate ligase activation, has remained unclear. Here, Lenka et al. use various truncation mutants of Parkin in an attempt to understand the likelihood of dimerization (in support of an "in trans" model for catalysis).
Strengths:
The results are bolstered by several distinct approaches including analytical SEC with cleavable Parkin constructs, ITC interaction studies, ubiquitination assays, protein crystallography, and cellular localization studies.
Weaknesses:
As presented, however, the storyline is very confusing to follow and several lines of experimentation felt like distractions from the primary message. Furthermore, many experiments could only indirectly support the author's conclusions, and therefore the final picture of what new features can be firmly added to the model of Parkin activation and function is unclear.
Following peer review and revision, the claims are still not fully supported by direct evidence. While the experimental system may be necessary and/or convenient given the unique challenges in studying Parkin, it does not directly speak toward the conclusions that the authors make, nor does it provide an accurate representation of biology.
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Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review):
Summary:
The authors used structural and biophysical methods to provide insight into Parkin regulation. The breadth of data supporting their findings was impressive and generally well-orchestrated. Still, the impact of their results builds on recent structural studies and the stated impact is based on these prior works.
Strengths:
(1) After reading through the paper, the major findings are:
- RING2 and pUbl compete for binding to RING0.
- Parkin can dimerize.
- ACT plays an important role in enzyme kinetics.
(2) The use of molecular scissors in their construct represents a creative approach to examining inter-domain interactions.
(3) From my assessment, the experiments are well-conceived and executed.
We thank the reviewer for their positive remark and extremely helpful suggestions.
Weaknesses:
The manuscript, as written, is NOT for a general audience. Admittedly, I am not an expert on Parkin structure and function, but I had to do a lot of homework to try to understand the underlying rationale and impact. This reflects, I think, that the work generally represents an incremental advance on recent structural findings.
To this point, it is hard to understand the impact of this work without more information highlighting the novelty. There are several structures of Parkin in various auto-inhibited states, and it was hard to delineate how this is different.
For the sake of the general audience, we have included all the details of Parkin structures and conformations seen (Extended Fig. 1). The structures in the present study are to validate the biophysical/biochemical experiments, highlighting key findings. For example, we solved the phospho-Parkin (complex with pUb) structure after treatment with 3C protease (Fig. 2C), which washes off the pUbl-linker, as shown in Fig 2B. The structure of the pUbl-linker depleted phospho-Parkin-pUb complex showed that RING2 returned to the closed state (Fig. 2C), which is confirmation of the SEC assay in Fig. 2B. Similarly, the structure of the pUbl-linker depleted phospho-Parkin R163D/K211N-pUb complex (Fig. 3C), was done to validate the SEC data showing displacement of pUbl-linker is independent of pUbl interaction with the basic patch on RING0 (Fig. 3B). In addition, the latter structure also revealed a new donor ubiquitin binding pocket in the linker (connecting REP and RING2) region of Parkin (Fig. 9). Similarly, trans-complex structure of phospho-Parkin (Fig. 4D) was done to validate the biophysical data (Fig. 4A-C, Fig. 5A-D) showing trans-complex between phospho-Parkin and native Parkin. The latter also confirmed that the trans-complex was mediated by interactions between pUbl and the basic patch on RING0 (Fig. 4D). Furthermore, we noticed that the ACT region was disordered in the trans-complex between phospho-Parkin (1-140 + 141-382 + pUb) (Fig. 8A) which had ACT from the trans molecule, indicating ACT might be present in the cis molecule. The latter was validated from the structure of trans-complex between phospho-Parkin with cis ACT (1-76 + 77-382 + pUb) (Fig. 8C), showing the ordered ACT region. The structural finding was further validated by biochemical assays (Fig. 8 D-F, Extended Data Fig. 9C-E).
The structure of TEV-treated R0RBR (TEV) (Extended Data Fig. 4C) was done to ensure that the inclusion of TEV and treatment with TEV protease did not perturb Parkin folding, an important control for our biophysical experiments.
As noted, I appreciated the use of protease sites in the fusion protein construct. It is unclear how the loop region might affect the protein structure and function. The authors worked to demonstrate that this did not introduce artifacts, but the biological context is missing.
We thank the reviewer for appreciating the use of protease sites in the fusion protein construct. Protease sites were used to overcome the competing mode of binding that makes interactions very transient and beyond the detection limit of methods such as ITC or SEC. While these interactions are quite transient in nature, they could still be useful for the activation of various Parkin isoforms that lack either the Ubl domain or RING2 domain (Extended Data Fig. 6, Fig. 10). Also, our Parkin localization assays also suggest an important role of these interactions in the recruitment of Parkin molecules to the damaged mitochondria (Fig. 6).
While it is likely that the binding is competitive between the Ubl and RING2 domains, the data is not quantitative. Is it known whether the folding of the distinct domains is independent? Or are there interactions that alter folding? It seems plausible that conformational rearrangements may invoke an orientation of domains that would be incompatible. The biological context for the importance of this interaction was not clear to me.
This is a great point. In the revised manuscript, we have included quantitative data between phospho-Parkin and untethered ∆Ubl-Parkin (TEV) (Fig. 5B) showing similar interactions using phospho-Parkin K211N and untethered ∆Ubl-Parkin (TEV) (Fig. 4B). Folding of Ubl domain or various combinations of RING domains lacking Ubl seems okay. Also, folding of the RING2 domain on its own appears to be fine. However, human Parkin lacking the RING2 domain seems to have some folding issues, majorly due to exposure of hydrophobic pocket on RING0, also suggested by previous efforts (Gladkova et al.ref. 24, Sauve et al. ref. 29). The latter could be overcome by co-expression of RING2 lacking Parkin construct with PINK1 (Sauve et al. ref. 29) as phospho-Ubl binds on the same hydrophobic pocket on RING0 where RING2 binds. A drastic reduction in the melting temperature of phospho-Parkin (Gladkova et al.ref. 24), very likely due to exposure of hydrophobic surface between RING0 and RING2, correlates with the folding issues of RING0 exposed human Parkin constructs.
From the biological context, the competing nature between phospho-Ubl and RING2 domains could block the non-specific interaction of phosphorylated-ubiquitin-like proteins (phospho-Ub or phospho-NEDD8) with RING0 (Lenka et al. ref. 33), during Parkin activation.
(5) What is the rationale for mutating Lys211 to Asn? Were other mutations tried? Glu? Ala? Just missing the rationale. I think this may have been identified previously in the field, but not clear what this mutation represents biologically.
Lys211Asn is a Parkinson’s disease mutation; therefore, we decided to use the same mutation for biophysical studies.
I was confused about how the phospho-proteins were generated. After looking through the methods, there appear to be phosphorylation experiments, but it is unclear what the efficiency was for each protein (i.e. what % gets modified). In the text, the authors refer to phospho-Parkin (T270R, C431A), but not clear how these mutations might influence this process. I gather that these are catalytically inactive, but it is unclear to me how this is catalyzing the ubiquitination in the assay.
This is an excellent question. Because different phosphorylation statuses would affect the analysis, we ensured complete phosphorylation status using Phos-Tag SDS-PAGE, as shown below.
Author response image 1.
Our biophysical experiments in Fig. 5C show that trans complex formation is mediated by interactions between the basic patch (comprising K161, R163, K211) on RING0 and phospho-Ubl domain in trans. These interactions result in the displacement of RING2 (Fig. 5C). Parkin activation is mediated by displacement of RING2 and exposure of catalytic C431 on RING2. While phospho-Parkin T270R/C431A is catalytically dead, the phospho-Ubl domain of phospho-Parkin T270R/C431would bind to the basic patch on RING0 of WT-Parkin resulting in activation of WT-Parkin as shown in Fig. 5E. A schematic figure is shown below to explain the same.
Author response image 2.
(7) The authors note that "ACT can be complemented in trans; however, it is more efficient in cis", but it is unclear whether both would be important or if the favored interaction is dominant in a biological context.
First, this is an excellent question about the biological context of ACT and needs further exploration. While due to the flexible nature of ACT, it can be complemented both in cis and trans, we can only speculate cis interactions between ACT and RING0 could be more relevant from the biological context as during protein synthesis and folding, ACT would be translated before RING2, and thus ACT would occupy the small hydrophobic patch on RING0 in cis. Unpublished data shows the replacement of the ACT region by Biogen compounds to activate Parkin (https://doi.org/10.21203/rs.3.rs-4119143/v1). The latter finding further suggests the flexibility in this region.
(8) The authors repeatedly note that this study could aid in the development of small-molecule regulators against Parkin to treat PD, but this is a long way off. And it is not clear from their manuscript how this would be achieved. As stated, this is conjecture.
As suggested by this reviewer, we have removed this point in the revised manuscript.
Reviewer #2 (Public Review):
This manuscript uses biochemistry and X-ray crystallography to further probe the molecular mechanism of Parkin regulation and activation. Using a construct that incorporates cleavage sites between different Parkin domains to increase the local concentration of specific domains (i.e., molecular scissors), the authors suggest that competitive binding between the p-Ubl and RING2 domains for the RING0 domain regulates Parkin activity. Further, they demonstrate that this competition can occur in trans, with a p-Ubl domain of one Parkin molecule binding the RING0 domain of a second monomer, thus activating the catalytic RING1 domain. In addition, they suggest that the ACT domain can similarly bind and activate Parkin in trans, albeit at a lower efficiency than that observed for p-Ubl. The authors also suggest from crystal structure analysis and some biochemical experiments that the linker region between RING2 and repressor elements interacts with the donor ubiquitin to enhance Parkin activity.<br /> Ultimately this manuscript challenges previous work suggesting that the p-Ubl domain does not bind to the Parkin core in the mechanism of Parkin activation. The use of the 'molecular scissors' approach to probe these effects is an interesting approach to probe this type of competitive binding. However, there are issues with the experimental approach manuscript that detract from the overall quality and potential impact of the work.
We thank the reviewer for their positive remark and constructive suggestions.
The competitive binding between p-Ubl and RING2 domains for the Parkin core could have been better defined using biophysical and biochemical approaches that explicitly define the relative affinities that dictate these interactions. A better understanding of these affinities could provide more insight into the relative bindings of these domains, especially as it relates to the in trans interactions.
This is an excellent point regarding the relative affinities of pUbl and RING2 for the Parkin core (lacking Ubl and RING2). While we could purify p-Ubl, we failed to purify human Parkin (lacking RING2 and phospho-Ubl). The latter folding issues were likely due to the exposure of a highly hydrophobic surface on RING0 (as shown below) in the absence of pUbl and RING2 in the R0RB construct. Also, RING2 with an exposed hydrophobic surface would be prone to folding issues, which is not suitable for affinity measurements. A drastic reduction in the melting temperature of phospho-Parkin (Gladkova et al.ref. 24) also highlights the importance of a hydrophobic surface between RING0 and RING2 on Parkin folding/stability. A separate study would be required to try these Parkin constructs from different species and ensure proper folding before using them for affinity measurements.
Author response image 3.
I also have concerns about the results of using molecular scissors to 'increase local concentrations' and allow for binding to be observed. These experiments are done primarily using proteolytic cleavage of different domains followed by size exclusion chromatography. ITC experiments suggest that the binding constants for these interactions are in the µM range, although these experiments are problematic as the authors indicate in the text that protein precipitation was observed during these experiments. This type of binding could easily be measured in other assays. My issue relates to the ability of a protein complex (comprising the core and cleaved domains) with a Kd of 1 µM to be maintained in an SEC experiment. The off-rates for these complexes must be exceeding slow, which doesn't really correspond to the low µM binding constants discussed in the text. How do the authors explain this? What is driving the Koff to levels sufficiently slow to prevent dissociation by SEC? Considering that the authors are challenging previous work describing the lack of binding between the p-Ubl domain and the core, these issues should be better resolved in this current manuscript. Further, it's important to have a more detailed understanding of relative affinities when considering the functional implications of this competition in the context of full-length Parkin. Similar comments could be made about the ACT experiments described in the text.
This is a great point. In the revised manuscript, we repeated ITC measurements in a different buffer system, which gave nice ITC data. In the revised manuscript, we have also performed ITC measurements using native phospho-Parkin. Phospho-Parkin and untethered ∆Ubl-Parkin (TEV) (Fig. 5B) show similar affinities as seen between phospho-Parkin K211N and untethered ∆Ubl-Parkin (TEV) (Fig. 4B). However, Kd values were consistent in the range of 1.0 ± 0.4 µM which could not address the reviewer’s point regarding slow off-rate. The crystal structure of the trans-complex of phospho-Parkin shows several hydrophobic and ionic interactions between p-Ubl and Parkin core, suggesting a strong interaction and, thus, justifying the co-elution on SEC. Additionally, ITC measurements between E2-Ub and P-Parkin-pUb show similar affinity (Kd = 0.9 ± 0.2 µM) (Kumar et al., 2015, EMBO J.), and yet they co-elute on SEC (Kumar et al., 2015, EMBO J.).
Ultimately, this work does suggest additional insights into the mechanism of Parkin activation that could contribute to the field. There is a lot of information included in this manuscript, giving it breadth, albeit at the cost of depth for the study of specific interactions. Further, I felt that the authors oversold some of their data in the text, and I'd recommend being a bit more careful when claiming an experiment 'confirms' a specific model. In many cases, there are other models that could explain similar results. For example, in Figure 1C, the authors state that their crystal structure 'confirms' that "RING2 is transiently displaced from the RING0 domain and returns to its original position after washing off the p-Ubl linker". However, it isn't clear to me that RING2 ever dissociated when prepared this way. While there are issues with the work that I feel should be further addressed with additional experiments, there are interesting mechanistic details suggested by this work that could improve our understanding of Parkin activation. However, the full impact of this work won't be fully appreciated until there is a more thorough understanding of the regulation and competitive binding between p-Ubl and RIGN2 to RORB both in cis and in trans.
We thank the reviewer for their positive comment. In the revised manuscript, we have included the reviewer’s suggestion. The conformational changes in phospho-Parkin were established from the SEC assay (Fig. 2A and Fig. 2B), which show displacement/association of phospho-Ubl or RING2 after treatment of phospho-Parkin with 3C and TEV, respectively. For crystallization, we first phosphorylated Parkin, where RING2 is displaced due to phospho-Ubl (as shown in SEC), followed by treatment with 3C protease, which led to pUbl wash-off. The Parkin core separated from phospho-Ubl on SEC was used for crystallization and structure determination in Fig. 2C, where RING2 returned to the RING0 pocket, which confirms SEC data (Fig. 2B).
Reviewer #3 (Public Review):
Summary:
In their manuscript "Additional feedforward mechanism of Parkin activation via binding of phospho-UBL and RING0 in trans", Lenka et al present data that could suggest an "in trans" model of Parkin ubiquitination activity. Parkin is an intensely studied E3 ligase implicated in mitophagy, whereby missense mutations to the PARK2 gene are known to cause autosomal recessive juvenile parkinsonism. From a mechanistic point of view, Parkin is extremely complex. Its activity is tightly controlled by several modes of auto-inhibition that must be released by queues of mitochondrial damage. While the general overview of Parkin activation has been mapped out in recent years, several details have remained murky. In particular, whether Parkin dimerizes as part of its feed-forward signaling mechanism, and whether said dimerization can facilitate ligase activation, has remained unclear. Here, Lenka et al. use various truncation mutants of Parkin in an attempt to understand the likelihood of dimerization (in support of an "in trans" model for catalysis).
Strengths:
The results are bolstered by several distinct approaches including analytical SEC with cleavable Parkin constructs, ITC interaction studies, ubiquitination assays, protein crystallography, and cellular localization studies.
We thank the reviewer for their positive remark.
Weaknesses:
As presented, however, the storyline is very confusing to follow and several lines of experimentation felt like distractions from the primary message. Furthermore, many experiments could only indirectly support the author's conclusions, and therefore the final picture of what new features can be firmly added to the model of Parkin activation and function is unclear.
We thank the reviewer for their constructive criticism, which has helped us to improve the quality of this manuscript.
Major concerns:
(1) This manuscript solves numerous crystal structures of various Parkin components to help support their idea of in trans transfer. The way these structures are presented more resemble models and it is unclear from the figures that these are new complexes solved in this work, and what new insights can be gleaned from them.
The structures in the present study are to validate the biophysical/biochemical experiments highlighting key findings. For example, we solved the phospho-Parkin (complex with pUb) structure after treatment with 3C protease (Fig. 2C), which washes off the pUbl-linker, as shown in Fig. 2B. The structure of pUbl-linker depleted phospho-Parkin-pUb complex showed that RING2 returned to the closed state (Fig. 2C), which is confirmation of the SEC assay in Fig. 2B. Similarly, the structure of the pUbl-linker depleted phospho-Parkin R163D/K211N-pUb complex (Fig. 3C), was done to validate the SEC data showing displacement of pUbl-linker is independent of pUbl interaction with the basic patch on RING0 (Fig. 3B). In addition, the latter structure also revealed a new donor ubiquitin binding pocket in the linker (connecting REP and RING2) region of Parkin (Fig. 9). Similarly, trans-complex structure of phospho-Parkin (Fig. 4D) was done to validate the biophysical data (Fig. 4A-C, Fig. 5A-D) showing trans-complex between phospho-Parkin and native Parkin. The latter also confirmed that the trans-complex was mediated by interactions between pUbl and the basic patch on RING0 (Fig. 4D). Furthermore, we noticed that the ACT region was disordered in the trans-complex between phospho-Parkin (1-140 + 141-382 + pUb) (Fig. 8A) which had ACT from the trans molecule, indicating ACT might be present in the cis molecule. The latter was validated from the structure of trans-complex between phospho-Parkin with cis ACT (1-76 + 77-382 + pUb) (Fig. 8C), showing the ordered ACT region. The structural finding was further validated by biochemical assays (Fig. 8 D-F, Extended Data Fig. 9C-E).
The structure of TEV-treated R0RBR (TEV) (Extended Data Fig. 4C) was done to ensure that the inclusion of TEV and treatment with TEV protease did not perturb Parkin folding, an important control for our biophysical experiments.
(2) There are no experiments that definitively show the in trans activation of Parkin. The binding experiments and size exclusion chromatography are a good start, but the way these experiments are performed, they'd be better suited as support for a stronger experiment showing Parkin dimerization. In addition, the rationale for an in trans activation model is not convincingly explained until the concept of Parkin isoforms is introduced in the Discussion. The authors should consider expanding this concept into other parts of the manuscript.
We thank the reviewer for appreciating the Parkin dimerization. Our biophysical data in Fig. 5C shows that Parkin dimerization is mediated by interactions between phospho-Ubl and RING0 in trans, leading to the displacement of RING2. However, Parkin K211N (on RING0) mutation perturbs interaction with phospho-Parkin and leads to loss of Parkin dimerization and loss of RING2 displacement (Fig. 5C). The interaction between pUbl and K211 pocket on RING0 leads to the displacement of RING2 resulting in Parkin activation as catalytic residue C431 on RING2 is exposed for catalysis. The biophysical experiment is further confirmed by a biochemical experiment where the addition of catalytically in-active phospho-Parkin T270R/C431A activates autoinhibited WT-Parkin in trans using the mechanism as discussed (a schematic representation also shown in Author response image 2).
We thank this reviewer regarding Parkin isoforms. In the revised manuscript, we have included Parkin isoforms in the results section, too.
(2a) For the in trans activation experiment using wt Parkin and pParkin (T270R/C431A) (Figure 3D), there needs to be a large excess of pParkin to stimulate the catalytic activity of wt Parkin. This experiment has low cellular relevance as these point mutations are unlikely to occur together to create this nonfunctional pParkin protein. In the case of pParkin activating wt Parkin (regardless of artificial point mutations inserted to study specifically the in trans activation), if there needs to be much more pParkin around to fully activate wt Parkin, isn't it just more likely that the pParkin would activate in cis?
To test phospho-Parkin as an activator of Parkin in trans, we wanted to use the catalytically inactive version of phospho-Parkin to avoid the background activity of p-Parkin. While it is true that a large excess of pParkin (T270R/C431A) is required to activate WT-Parkin in the in vitro set-up, it is not very surprising as in WT-Parkin, the unphosphorylated Ubl domain would block the E2 binding site on RING1. Also, due to interactions between pParkin (T270R/C431A) molecules, the net concentration of pParkin (T270R/C431A) as an activator would be much lower. However, the Ubl blocking E2 binding site on RING1 won’t be an issue between phospho-Parkin molecules or between Parkin isoforms (lacking Ubl domain or RING2).
(2ai) Another underlying issue with this experiment is that the authors do not consider the possibility that the increased activity observed is a result of increased "substrate" for auto-ubiquitination, as opposed to any role in catalytic activation. Have the authors considered looking at Miro as a substrate in order to control for this?
This is quite an interesting point. However, this will be only possible if Parkin is ubiquitinated in trans, as auto-ubiquitination is possible with active Parkin and not with catalytically dead (phospho-Parkin T270R, C431A) or autoinhibited (WT-Parkin). Also, in the previous version of the manuscript, where we used only phospho-Ubl as an activator of Parkin in trans, we tested Miro1 ubiquitination and auto-ubiquitination, and the results were the same (Author response image 4).
Author response image 4.
(2b) The authors mention a "higher net concentration" of the "fused domains" with RING0, and use this to justify artificially cleaving the Ubl or RING2 domains from the Parkin core. This fact should be moot. In cells, it is expected there will only be a 1:1 ratio of the Parkin core with the Ubl or RING2 domains. To date, there is no evidence suggesting multiple pUbls or multiple RING2s can bind the RING0 binding site. In fact, the authors here even show that either the RING2 or pUbl needs to be displaced to permit the binding of the other domain. That being said, there would be no "higher net concentration" because there would always be the same molar equivalents of Ubl, RING2, and the Parkin core.
We apologize for the confusion. “Higher net concentration” is with respect to fused domains versus the domain provided in trans. Due to the competing nature of the interactions between pUbl/RING2 and RING0, the interactions are too transient and beyond the detection limit of the biophysical techniques. While the domains are fused (for example, RING0-RING2 in the same polypeptide) in a polypeptide, their effective concentrations are much higher than those (for example, pUbl) provided in trans; thus, biophysical methods fail to detect the interaction. Treatment with protease solves the above issue due to the higher net concentration of the fused domain, and trans interactions can be measured using biophysical techniques. However, the nature of these interactions and conformational changes is very transient, which is also suggested by the data. Therefore, Parkin molecules will never remain associated; rather, Parkin will transiently interact and activate Parkin molecules in trans.
(2c) A larger issue remaining in terms of Parkin activation is the lack of clarity surrounding the role of the linker (77-140); particularly whether its primary role is to tether the Ubl to the cis Parkin molecule versus a role in permitting distal interactions to a trans molecule. The way the authors have conducted the experiments presented in Figure 2 limits the possible interactions that the activated pUbl could have by (a) ablating the binding site in the cis molecule with the K211N mutation; (b) further blocking the binding site in the cis molecule by keeping the RING2 domain intact. These restrictions to the cis parkin molecule effectively force the pUbl to bind in trans. A competition experiment to demonstrate the likelihood of cis or trans activation in direct comparison with each other would provide stronger evidence for trans activation.
This is an excellent point. In the revised manuscript, we have performed experiments using native phospho-Parkin (Revised Figure 5), and the results are consistent with those in Figure 2 ( Revised Figure 4), where we used the K211N mutation.
(3) A major limitation of this study is that the authors interpret structural flexibility from experiments that do not report directly on flexibility. The analytical SEC experiments report on binding affinity and more specifically off-rates. By removing the interdomain linkages, the accompanying on-rate would be drastically impacted, and thus the observations are disconnected from a native scenario. Likewise, observations from protein crystallography can be consistent with flexibility, but certainly should not be directly interpreted in this manner. Rigorous determination of linker and/or domain flexibility would require alternative methods that measure this directly.
We also agree with the reviewer that these methods do not directly capture structural flexibility. Also, rigorous determination of linker flexibility would require alternative methods that measure this directly. However, due to the complex nature of interactions and technical limitations, breaking the interdomain linkages was the best possible way to capture interactions in trans. Interestingly, all previous methods that report cis interactions between pUbl and RING0 also used a similar approach (Gladkova et al.ref. 24, Sauve et al. ref. 29).
(4) The analysis of the ACT element comes across as incomplete. The authors make a point of a competing interaction with Lys48 of the Ubl domain, but the significance of this is unclear. It is possible that this observation could be an overinterpretation of the crystal structures. Additionally, the rationale for why the ACT element should or shouldn't contribute to in trans activation of different Parkin constructs is not clear. Lastly, the conclusion that this work explains the evolutionary nature of this element in chordates is highly overstated.
We agree with the reviewer that the significance of Lys48 is unclear. We have presented this just as one of the observations from the crystal structure. As the reviewer suggested, we have removed the sentence about the evolutionary nature of this element from the revised manuscript.
(5) The analysis of the REP linker element also seems incomplete. The authors identify contacts to a neighboring pUb molecule in their crystal structure, but the connection between this interface (which could be a crystallization artifact) and their biochemical activity data is not straightforward. The analysis of flexibility within this region using crystallographic and AlphaFold modeling observations is very indirect. The authors also draw parallels with linker regions in other RBR ligases that are involved in recognizing the E2-loaded Ub. Firstly, it is not clear from the text or figures whether the "conserved" hydrophobic within the linker region is involved in these alternative Ub interfaces. And secondly, the authors appear to jump to the conclusion that the Parkin linker region also binds an E2-loaded Ub, even though their original observation from the crystal structure seems inconsistent with this. The entire analysis feels very preliminary and also comes across as tangential to the primary storyline of in trans Parkin activation.
We agree with the reviewer that crystal structure data and biochemical data are not directly linked. In the revised manuscript, we have also highlighted the conserved hydrophobic in the linker region at the ubiquitin interface (Fig. 9C and Extended Data Fig. 11A), which was somehow missed in the original manuscript. We want to add that a very similar analysis and supporting experiments identified donor ubiquitin-binding sites on the IBR and helix connecting RING1-IBR (Kumar et al., Nature Str. and Mol. Biol., 2017), which several other groups later confirmed. In the mentioned study, the Ubl domain of Parkin from the symmetry mate Parkin molecule was identified as a mimic of “donor ubiquitin” on IBR and helix connecting RING1-IBR.
In the present study, a neighboring pUb molecule in the crystal structure is identified as a donor ubiquitin mimic (Fig. 9C) by supporting biophysical/biochemical experiments. First, we show that mutation of I411A in the REP linker of Parkin perturbs Parkin interaction with E2~Ub (donor) (Fig. 9F). Another supporting experiment was performed using a Ubiquitin-VS probe assay, which is independent of E2. Assays using Ubiquitin-VS show that I411A mutation in the REP-RING2 linker perturbs Parkin charging with Ubiquitin-VS (Extended Data Fig. 11 B). Furthermore, the biophysical data showing loss of Parkin interaction with donor ubiquitin is further supported by ubiquitination assays. Mutations in the REP-RING2 linker perturb the Parkin activity (Fig. 9E), confirming biophysical data. This is further confirmed by mutations (L71A or L73A) on ubiquitin (Extended Data Fig. 11C), resulting in loss of Parkin activity. The above experiments nicely establish the role of the REP-RING2 linker in interaction with donor ubiquitin, which is consistent with other RBRs (Extended Data Fig. 11A).
While we agree with the reviewer that this appears tangential to the primary storyline in trans-Parkin activation, we decided to include this data because it could be of interest to the field.
Recommendations for the authors:
Reviewer #1 (Recommendations For The Authors):
(1) For clarity, a schematic of the domain architecture of Parkin would be helpful at the outset in the main figures. This will help with the introduction to better understand the protein organization. This is lost in the Extended Figure in my opinion.
We thank the reviewer for suggesting this, which we have included in Figure 1 of the revised manuscript.
(2) Related to the competition between the Ubl and RING2 domains, can competition be shown through another method? SPR, ITC, etc? ITC was used in other experiments, but only in the context of mutations (Lys211Asn)? Can this be done with WT sequence?
This is an excellent suggestion. In the revised Figure 5, we have performed ITC experiment using WT Parkin, and the results are consistent with what we observed using Lys211Asn Parkin.
(3) The authors also note that "the AlphaFold model shows a helical structure in the linker region of Parkin (Extended Data Figure 10C), further confirming the flexible nature of this region"... but the secondary structure would not be inherently flexible. This is confusing.
The flexibility is in terms of the conformation of this linker region observed under the open or closed state of Parkin. In the revised manuscript, we have explained this point more clearly.
(4) The manuscript needs extensive revision to improve its readability. Minor grammatical mistakes were prevalent throughout.
We thank the reviewer for pointing out this and we have corrected these in the revised manuscript.
(5) The confocal images are nice, but inset panels may help highlight the regions of interest (ROIs).
This is corrected in the revised manuscript.
(6) Trans is misspelled ("tans") towards the end of the second paragraph on page 16.
This is corrected in the revised manuscript.
(7) The schematics are helpful, but some of the lettering in Figure 2 is very small.
This is corrected in the revised manuscript.
Reviewer #3 (Recommendations For The Authors):
(1) A significant portion of the results section refers to the supplement, making the overall readability very difficult.
We accept this issue as a lot of relevant data could not be added to the main figures and thus ended up in the supplement. In the revised manuscript, we have moved some of the supplementary figures to the main figures.
(2) Interpretation of the experiments utilizing many different Parkin constructs and cleavage scenarios (particularly the SEC and crystallography experiments) is extremely difficult. The work would benefit from a layout of the Parkin model system, highlighting cleavage sites, key domain terminology, and mutations used in the study, presented together and early on in the manuscript. Using this to identify a simpler system of referencing Parkin constructs would also be a large improvement.
This is a great suggestion. We have included these points in the revised manuscript, which has improved the readability.
(3) Lines 81-83; the authors say they "demonstrate the conformational changes in Parkin during the activation process", but fail to show any actual conformational changes. Further, much of what is demonstrated in this work (in terms of crystal structures) corroborates existing literature. The authors should use caution not to overstate their original conclusions in light of the large body of work in this area.
We thank the reviewer for pointing out this. We have corrected the above statement in the revised manuscript to indicate that we meant it in the context of trans conformational changes.
(4) Line 446 and 434; there is a discrepancy about which amino acid is present at residue 409. Is this a K408 typo? The authors also present mutational work on K416, but this residue is not shown in the structure panel.
We thank the reviewer for pointing out this. In the revised manuscript, we have corrected these typos.
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eLife assessment
This study presents important findings on the different polymorphs of alpha-synuclein filaments that form at various pH's during in vitro assembly reactions with purified recombinant protein. Of particular note is the discovery of two new polymorphs (1M and 5A) that form in PBS buffer at pH 7. The strength of the evidence presented is convincing. The work will be of interest to biochemists and biophysicists working on protein aggregation and amyloids.
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Reviewer #2 (Public Review):
Summary:
This is an exciting paper that explores the in vitro assembly of recombinant alpha-synuclein into amyloid filaments. The authors changed the pH and the composition of the assembly buffers, as well as the presence of different types of seeds, and analysed the resulting structures by cryo-EM.
Strengths:
By doing experiments at different pHs, the authors found that so-called type 2 and type-3 polymorphs form in a pH dependent manner. In addition, they find that type-1 filaments form in the presence of phosphate ions. One of their in vitro assembled type-1 polymorphs is similar to the alpha-synuclein filaments that were extracted from the brain of an individual with juvenile-onset synucleinopathy (JOS). They hypothesize that additional densities in a similar place as additional densities in the JOS fold correspond to phosphate ions.
Comments on the revised version:
This is OK now. I thank the authors for their constructive engagement with my comments.
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Reviewer #3 (Public Review):
Summary
The high heterogeneity nature of α-synuclein (α-syn) fibrils posed significant challenges in structural reconstruction of the ex vivo conformation. A deeper understanding of the factors influencing the formation of various α-syn polymorphs remains elusive. The manuscript by Frey et al. provides a comprehensive exploration of how pH variations (ranging from 5.8 to 7.4) affect the selection of α-syn polymorphs (specifically, Type1, 2 and 3) in vitro by using cryo-electron microscopy (cryo-EM) and helical reconstruction techniques. Crucially, the authors identify two novel polymorphs at pH 7.0 in PBS. These polymorphs bear resemblance to the structure of patient-derived juvenile-onset synucleinopathy (JOS) polymorph and diseased tissue amplified α-syn fibrils. The revised manuscript more strongly supports the notion that seeding is a non-polymorph-specific in the context of secondary nucleation-dominated aggregation, underscoring the irreplaceable role of pH in polymorph formation.
Strengths
This study systematically investigates the effects of environmental conditions and seeding on the structure of α-syn fibrils. It emphasizes the significant influence of environmental factors, especially pH, in determining the selection of α-syn polymorphs. The high-resolution structures obtained through cryo-EM enable a clear characterization of the composition and proportion of each polymorph in the sample. Collectively, this work provides a strong support for the pronounced sensitivity of α-syn fibril structures to the environmental conditions and systematically categorizes previously reported α-syn fibril structures. Furthermore, the identification of JOS-like polymorph also demonstrates the possibility of in vitro reconstruction of brain-derived α-syn fibril structures.
Weaknesses
All my previous concerns have been resolved to my satisfaction.
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Author response:
The following is the authors’ response to the previous reviews.
Revisions Round 1
Reviewer #1
We thank the reviewer for their careful reading of our manuscript and have taken all of their grammatical corrections into account.
Reviewer #2 (Public Review):
Weaknesses:
The paper contains multiple instances of non-scientific language, as indicated below. It would also benefit from additional details on the cryo-EM structure determination in the Methods and inclusion of commonly accepted requirements for cryo-EM structures, like examples of 2D class averages, raw micrographs, and FSC curves (between half-maps as well as between rigid-body fitted (or refined) atomic models of the different polymorphs and their corresponding maps). In addition, cryo-EM maps for the control experiments F1 and F2 should be presented in Figure 9.
We tried to correct the non-scientific language and have included the suggested data on the Cryo-EM analyses including new Figures 11-17. We did not collect data on the sample used for the seeds in the cross seeding experiments because we had already confirmed in multiple datasets that the conditions in F1 and F2 reproducibly produce fibrils of Type 1 and Type 3, respectively. We have now analyzed cryo-EM data for 6 more samples at pH 7.0 and found that several kinds of polymorphs (Types 1A, 1M, 2A, 2B and 5) are accessible at this pH, however the Type 3 polymorphs are not formed at pH 7.0 under the conditions that we used for aggregation.
Reviewer #2 (Recommendations For The Authors):
- remove unscientific language: "it seems that there are about as many unique atomic-resolution structures of these aggregates as there are publications describing them"
We have rephrased this sentence.
- for same reason, remove "Obviously, "
Done
- What does this mean? “polymorph-unspecific”
Rephrased as non-polymorph-specific
- What does this mean? "shallow amyloid energy hypersurface"
By “shallow hypersurface” we mean that the minimum of the multi-dimensional function that describes the energy of the amyloid is not so deep that subtle changes to the environment will not favor another fold/energy minimum. We have left the sentence because while it may not be perfect, it is concise and seems to get the point across.
- "The results also confirm the possibility of producing disease-relevant structure in vitro." -> This is incorrect as no disease-relevant structure was replicated in this work. Use another word like “suggest”.
We have changed to “suggest” as suggested.
- Remove "historically"
Done
- Rephrase “It has long been understood that all amyloids contain a common structural scaffold”
Changed to “It has long been established that all amyloids contain a common structural scaffold..”
- "Amyloid polymorphs whose differences lie in both their tertiary structure (the arrangement of the beta-strands) and the quaternary structure (protofilamentprotofilament assembly) have been found to display distinct biological activities [8]" -> I don't think this is true, different biological activities of amyloids have never been linked to their distinct structures.
We have added 5 new references (8-12) to support this sentence.
- Reference 10 is a comment on reference 9; it should be removed. Instead, as for alpha-synuclein, all papers describing the tau structures should be included.
We have removed the reference, but feel that the addition of all Tau structure references is not merited in this manuscript since we are not comparing them.
- Rephrase: "is not always 100% faithful"
Removed “100%”
- What is pseudo-C2 symmetry? Do the authors mean pseudo 2_1 symmetry (ie a 2-start helical symmetry)?
Thank for pointing this out. We did indeed mean pseudo 21 helical symmetry.
- Re-phrase: "alpha-Syn's chameleon-like behavior"
We have removed this phrase.
- "In the case of alpha-Syn, the secondary nucleation mechanism is based on the interaction of the positively charged N-terminal region of monomeric alpha-Syn and the disordered, negatively charged C-terminal region of the alpha-Syn amyloid fibrils [54]" -> I would say the mechanisms of secondary nucleation are not that well understood yet, so one may want to tune this down a bit.
We have changed this to “mechanism has been proposed to be”
- The paragraphs describing experiments by others are better suited for a Discussion rather than a Results section. Perhaps re-organize this part?
We have left the text intact as we are using a Results and Discussion format.
- A lot of information about Image processing seems to be missing: what steps were performed after initial model generation?
We have added more details in the methods section on the EM data processing and model analysis.
- Figure 1: Where is Type 4 on the pH scale?
We have adjusted the Fig 1 legend to clarify that pH scale is only applicable to the structures presented in this manuscript.
- Figure 2: This might be better incorporated as a subpanel of Figure 1.
We agree that this figure is somewhat of a loner on its own and we only added it in order to avoid confusion with the somewhat inconsistent naming scheme used for the Type 1B structure. However, we prefer to leave it as a separate figure so that it does not get dilute the impact of figure 1.
- Figure 3: What is the extra density at the bottom of Type 3B from pH 5.8 samples 1 and 2. pH 5.8 + 50mM NaCl (but not pH 5.8 + 100 mM NaCl)? Could this be an indication of a local minimum and the pH 5.8 + 100 mM NaCl structure is correct? Or is this a real difference between 0/50mM NaCl and 100 mM NaCl?
We did not see the extra density to which the reviewer is referring, however the images used in this panel are the based on the output of 3D-classification which is more likely to produce more artifacts than a 3D refinement. With this in mind, we did not see any significant differences in the refined structures and therefore only deposited the better quality map and model for each of the polymorph types.
- Figure 3: To what extent is Type 3B of pH 6.5 still a mixture of different types? The density looks poor. In general, in the absence of more details about the cryo-EM maps, it is hard to assess the quality of the structures presented.
In order to improve the quality of the images in this panel, a more complete separation of the particles from each polymorph was achieved via the filament subset selection tool in RELION 5. In each case, an unbiased could be created from the 2D classes via the relion_helix_inimodel2D program, further supporting the coexistence of 4 polymorphs in the pH 6.5 sample. The particles were individually refined to produce the respective maps that are now used in this figure.
- Many references are incorrect, containing "Preprint at (20xx)" statements.
This has been corrected.
Reviewer #3 (Public Review):
Weaknesses:
(1) The authors reveal that both Type 1 monofilament fibril polymorph (reminiscent of JOSlike polymorph) and Type 5 polymorph (akin to tissue-amplified-like polymorph) can both form under the same condition. Additionally, this condition also fosters the formation of flat ribbon-like fibril across different batches. Notably, at pH 5.8, variations in experimental groups yield disparate abundance ratios between polymorph 3B and 3C, indicating a degree of instability in fibrillar formation. The variability would potentially pose challenges for replicability in subsequent research. In light of these situations, I propose the following recommendations:
(a) An explicit elucidation of the factors contributing to these divergent outcomes under similar experimental conditions is warranted. This should include an exploration of whether variations in purified protein batches are contributing factors to the observed heterogeneity.
We are in complete agreement that understanding the factors that lead to polymorph variability is of utmost importance (and was the impetus for the manuscript itself). However the number of variables to explore is overwhelming and we will continue to investigate this in our future research. Regarding the variability between batches of purified protein, we also think that this could be a factor in the polymorph variability observed for otherwise “identical” aggregation conditions, particularly at pH 7 where the largest variety of polymorphs have been observed. However, even variation between identical replicates (samples created from the same protein solution and simply aggregated simultaneously in separate tubes) can lead to different outcomes (see datasets 15 and 16 in the revised Table 1) suggesting that there are stochastic processes that can determine the outcome of an individual aggregation experiment. While our data still indicates that Type 1,2 and 3 polymorphs are strongly selected by pH, the selection between interface variants 3B vs. 3C and 2A vs. 2B might also be affected by protein purity. Our standard purification protocol produces a single band by coomassie-stained SDS-PAGE however minor truncations and other impurities below a few percent would go undetected and, given the proposed roles of the N and C-termini in secondary nucleation, could have a large effect on polymorph selection and seeding. In line with the reviewer’s comments we now include a batch number for each EM dataset. While no new conclusions can be drawn from the inclusion of this additional data, we feel that it is important to acknowledge the possible role of batch to batch variability.
(b) To enhance the robustness of the conclusions, additional replicates of the experiments under the same condition should be conducted, ideally a minimum of three times.
The pH 5.8 conditions that yield Type 3 fibrils has already been repeated several times in the original manuscript. Since the pH 7.4 conditions produce the most common a-Syn polymorph (Type 1A) and were produced twice in this manuscript (once as an unseeded and once as a cross-seeded fibrilization) we decided to focus on the intermediate condition where the most variability had been seen (pH 7.0). The revised table 1 now has 6 new datasets (11-16) representing 6 independent aggregations at pH 7.0 starting from two different protein purification batches. The results is that we now produce the type 2A/B polymorphs in three samples and in two of these samples we once again observed the type 1M polymorph. The other samples produced Type 1A or non-twisted fibrils.
(c) Further investigation into whether different polymorphs formed under the same buffer condition could lead to distinct toxicological and pathology effects would be a valuable addition to the study.
The correlation of toxicity with structure would in principle be interesting. However the Type 1 and Type 3 polymorphs formed at pH 5.8 and 7.4 are not likely to be biologically relevant. The pH 7 polymorphs (Type 5 and 1M) would be more interesting because they form under the same conditions and might be related to some disease relevant structures. Still, it is rare that a single polymorph appears at 7.0 (the Type 5 represented only 10-20% of the fibrils in the sample and the Type 1M also had unidentified double-filament fibrils in the sample). We plan to pursue this line of research and hope to include it in a future publication.
(2) The cross-seeding study presented in the manuscript demonstrates the pivotal role of pH conditions in dictating conformation. However, an intriguing aspect that emerges is the potential role of seed concentration in determining the resultant product structure. This raises a critical question: at what specific seed concentration does the determining factor for polymorph selection shift from pH condition to seed concentration? A methodological robust approach to address this should be conducted through a series of experiments across a range of seed concentrations. Such an approach could delineate a clear boundary at which seed concentration begins to predominantly dictate the conformation, as opposed to pH conditions. Incorporating this aspect into the study would not only clarify the interplay between seed concentration and pH conditions, but also add a fascinating dimension to the understanding of polymorph selection mechanisms.
A more complete analysis of the mechanisms of aggregation, including the effect of seed concentration and the resulting polymorph specificity of the process, are all very important for our understanding of the aggregation pathways of alpha-synuclein and are currently the topic of ongoing investigations in our lab.
Furthermore, the study prompts additional queries regarding the behavior of cross-seeding production under the same pH conditions when employing seeds of distinct conformation. Evidence from various studies, such as those involving E46K and G51D cross-seeding, suggests that seed structure plays a crucial role in dictating polymorph selection. A key question is whether these products consistently mirror the structure of their respective seeds.
We thank the reviewer for reminding us to cite these studies as a clear example of polymorph selection by cross-seeding. Unfortunately, it is not 100% clear from the G51D cross seeding manuscript (https://doi.org/10.1038/s41467-021-26433-2) what conditions were used in the cross-seeding since different conditions were used for the seedless wild-type and mutant aggregations… however it appears that the wildtype without seeds was Tris pH 7.5 (although at 37C the pH could have dropped to 7ish) and the cross-seeded wild-type was in Phosphate buffer at pH 7.0. In the E46K cross-seeding manuscript, it appears that pH 7.5 Tris was used for all fibrilizations (https://doi.org/10.1073/pnas.2012435118). In any event, both results point to the fact that at pH 7.0-7.5 under low-seed conditions (0.5%) the Type 4 polymorph can propagate in a seed specific manner.
(3) In the Results section of "The buffer environment can dictate polymorph during seeded nucleation", the authors reference previous cell biological and biochemical assays to support the polymorph-specific seeding of MSA and PD patients under the same buffer conditions. This discussion is juxtaposed with recent research that compares the in vivo biological activities of hPFF, ampLB as well as LB, particularly in terms of seeding activity and pathology. Notably, this research suggests that ampLB, rather than hPFF, can accurately model the key aspects of Lewy Body Diseases (LBD) (refer to: https://doi.org/10.1038/s41467-023-42705-5). The critical issue here is the need to reconcile the phenomena observed in vitro with those in in-vivo or in-cell models. Given the low seed concentration reported in these studies, it is imperative for the authors to provide a more detailed explanation as to why the possible similar conformation could lead to divergent pathologies, including differences in cell-type preference and seeding capability.
We thank the reviewer for bring this recent report to our attention. The findings that ampLB and hPFF have different PK digestion patterns and that only the former is able to model key aspects of Lewy Body disease are in support of the seed-specific nature of some types of alpha-synuclein aggregation. We have added this to the discussion regarding the significant role that seed type and seed conditions likely play in polymorph selection.
(4) In the Method section of "Image processing", the authors describe the helical reconstruction procedure, without mentioning much detail about the 3D reconstruction and refinement process. For the benefit of reproducibility and to facilitate a deeper understanding among readers, the authors should enrich this part to include more comprehensive information, akin to the level of detail found in similar studies (refer to: https://doi.org/10.1038/nature23002).
As also suggested by reviewer #2, we have now added more comprehensive information on the 3D reconstruction and refinement process.
(5) The abbreviation of amino acids should be unified. In the Results section "On the structural heterogeneity of Type 1 polymorphs", the amino acids are denoted using three-letter abbreviation. Conversely, in the same section under "On the structural heterogeneity of Type 2 and 3 structures", amino acids are abbreviated using the one-letter format. For clarity and consistency, it is essential that a standardized format for amino acid abbreviations be adopted throughout the manuscript.
That makes perfect sense and had been corrected.
Reviewing Editor:
After discussion among the reviewers, it was decided that point 2 in Reviewer #3's Public Review (about the experiments with different concentrations of seeds) would probably lie outside the scope of a reasonable revision for this work.
We agree as stated above and will continue to work on this important point.
Revisions Round 2
Reviewer #2 (Public Review):
I do worry that the FSC values of model-vs-map appear to be higher than expected from the corresponding FSCs between the half-maps (e.g. see Fig 13). The implication of this observation is that the atomic models may have been overfitted in the maps, which would have led to a deterioration of their geometry. A table with rmsd on bond lengths, angles, etc would probably show this. In addition, to check for overfitting, the atomic model for each data set could be refined in one of the half-maps, and then that same model could be used to calculate 2 FSC model-vs-map curves: one against the half-map it was refined in and one against the other half-map. Deviations between these two curves are an indication of overfitting.
Thank you for the recommendations for model validation. We have added the suggested statistics to Table 2 and performed the suggested model fitting to one of the half-maps and plotted 3 FSC model-vs-map curves: one for each half-map versus the model fit against only one half map and one for the model fit against the full map. We feel that the degree of overfitting is reasonable and does not significantly impact the quality of the models.
In addition, the sudden drop in the FSC curves in Figure 16 shows that something unexpected has happened to this refinement. Are the authors sure that only the procedures outlined in the Methods were used to create these curves? The unexpected nature of the FSC curve for this type (2A) raises doubts about the correctness of the reconstruction.
We thank the reviewer for the attention to detail. We should have caught this mistake. It turns out that in the last round of 3D refinement, the two half-maps become shifted with respect to each other in the z direction. We realigned the two maps using Chimera and then re-ran the postprocessing. The new maps have been deposited in EMD-50850. This mistake motivated us to inspect all of the maps and we found the same problem had occurred in the Type 3B maps. This was not noticed by the reviewer because we accidentally plotted the FSC curves from postprocessing from one refinement round before the one deposited in the EMD. We performed the same half-map shifting procedure for the Type 3B data and performed a final round of real-space refinement to produce new maps and models that have been deposited as EMD-50888 and 9FYP (superseding the previous entries).
Reviewer #3 (Public Review):
There are two minor points I recommend the authors to address:
(1) In the response to Weakness 1, point (3), the authors state that "the Type 5 represented only 10-20% of the fibrils in the sample." However, this information is not labeled in the corresponding Figure 4. I suggest the authors verify and label all relevant percentages in the figures to prevent misunderstandings.
We aim to be as transparent as possible and this information was included in the main text however we did not label the percentage of Type 5 fibrils in Figure 4 because that would make the other percentages ambiguous. The percentages in Figure 4 represent the ratio of helical segments used for each type of refined structure in the dataset (always adding up to 100%), not the percent of all fibrils in the dataset. That is, there are sometimes untwisted or unidentifiable fibrils in datasets and these were not accounted for in the listed percentages. We have added a sentence to the Figure 4 legend to explain to what the percentages refer.
(2) While the authors have detailed the helical reconstruction procedure in the Methods section, it is necessary to indicate the scale bar or box size in the figure legend of the 2D representative classes to ensure clarity and reproducibility.
Thank you for reminding us to add the scale bars. This is now done for the 2D classes in Figures 11-17.
Recommendations for the authors:
Reviewer #2 (Recommendations For The Authors):
A critical look at the maps and models of the various structures at this stage may prevent the authors from entering suboptimal structures into the databases.
We agree. Thank you for suggesting this.
Reviewer #3 (Recommendations For The Authors):
The authors have responded adequately to these critiques in the revised version of the manuscript. There are two minor points.
(1) The authors state that "the Type 5 represented only 10-20% of the fibrils in the sample." However, this information is not labeled in the corresponding Figure 4. I suggest the authors verify and label all relevant percentages in the figures to prevent misunderstandings.
(2) While the authors have detailed the helical reconstruction procedure in the Methods section, it is necessary to indicate the scale bar or box size in the figure legend of the 2D representative classes to ensure clarity and reproducibility.
Answered in public comments
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eLife assessment
This important study reports the molecular function of the SARS-CoV-2 helicase NSP13, which inhibits the transcriptional activity of the YAP/TEAD complex in vitro and in vivo. The evidence supporting the authors' claims is solid, with rigorous cell biological assays and multi-omic studies. This work will be of interest to scientists studying COVID-19 infection and the Hippo-YAP signaling pathway.
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Reviewer #1 (Public Review):
In the manuscript entitled "SARS-CoV-2 NSP13 interacts with TEAD to suppress Hippo-YAP signaling", Meng et al. report that SARS-CoV-2 infection disrupts YAP downstream gene transcription in both patient lung samples and the iPSC-cardiomyocytes. Among the tested SARS-CoV-2 proteins, the helicase nonstructural protein 13 (NSP13) was identified to target YAP transcriptional activity both in vitro and in vivo, independent of the Hippo pathway. Mechanistically, NSP13 inhibits YAP transcriptional activity through its interaction with TEAD4 and a group of nuclear repressor proteins, a process that requires its helicase activity. Overall, this study uncovers a novel regulation of the YAP/TEAD complex by SARS-CoV-2 infection, highlighting its impact on cellular signaling events. The manuscript is well-written and easy to follow. Here are some suggestions for the authors to further improve their work.
Major points
(1) The authors discovered a novel regulation of the Hippo-YAP pathway by SARS-CoV-2 infection but did not address the pathological significance of this finding. It remains unclear why YAP downstream gene transcription needs to be inhibited in response to SARS-CoV-2 infection. Is this inhibition crucial for the innate immune response to SARS-CoV-2? The authors should re-analyze their snRNA-seq and bulk RNA-seq data described in Figure 1 to determine whether any of the affected YAP downstream genes are involved in this process.
(2) The authors concluded that helicase activity is required for NSP13-induced inhibition of YAP transcriptional activity based on mutation studies (Figure 3B). This finding is somewhat confusing, as K131, K345/K347, and R567 are all essential residues for NSP13 helicase activity while mutating K131 did not affect NSP13's ability to inhibit YAP (Figure 3B). Additionally, there are no data showing exactly how NSP13 inhibits the YAP/TEAD complex through its helicase function. This point was also not reflected in their proposed working model (Figure 4H).
(3) The proposed model that NSP13 binds TEAD4 to recruit repressor proteins and inhibits YAP/TEAD downstream gene transcription (Figure 4H) needs further characterization. First, it is notable that the provided NSP13 IP-MS data did not reveal any TEAD family members as binding proteins for NSP13 (Supplement Figure 4C and the tables), suggesting that NSP13 may modulate the YAP/TEAD complex through other mechanisms, possibly involving other binding proteins. Second, NSP13 is a DNA-binding protein, and its nucleic acid-binding mutant K345A/K347A failed to inhibit YAP transcriptional activity (Figure 3B). The authors should investigate whether NSP13 could bind to the TEAD binding sequence or the nearby sequence on the genome to modulate TEAD's DNA binding ability. Third, regarding the identified nuclear repressors, the authors should validate the interaction of NSP13 with the ones whose loss activates YAP transcriptional activity (Figure 4G). Lastly, why can't NSP13 bind TEAD4 in the cytoplasmic fractionation if both NSP13 and TEAD4 are detected there (Figure 3B)? This finding indicates their interaction is not a direct protein-protein interaction but is mediated by something in the nucleus, such as genomic DNA.
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